Novel block copolymer and micelle compositions and methods of use thereof

ABSTRACT

wherein R′ is —H or —CH3, wherein R is —NR1R2, wherein R1 and R2 are alkyl groups, wherein R1 and R2 are the same or different, wherein R1 and R2 together have from 5 to 16 carbons, wherein R1 and R2 may optionally join to form a ring, wherein n is 1 to about 10, and wherein x is about 20 to about 200 in total. Also provided are pH-sensitive micelle compositions for therapeutic and diagnostic applications.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumbersCA129011, CA102792 and EB005394 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Multifunctional nanoparticles have received attention in a wide range ofapplications such as biosensors, diagnostic nanoprobes and targeted drugdelivery systems. These efforts have been driven to a large extent bythe need to improve biological specificity with reduced side effects indiagnosis and therapy through the precise, spatiotemporal control ofagent delivery in various physiological systems. In order to achievethis goal, efforts have been dedicated to develop stimuli-responsivenanoplatforms. Environmental stimuli that have been exploited forpinpointing the delivery efficiency include pH, temperature, enzymaticexpression, redox reaction and light induction. Among these activatingsignals, pH trigger is one of the most extensively studied stimuli basedon two types of pH differences: (a) pathological (e.g. tumor) vs. normaltissues and (b) acidic intracellular compartments.

For example, due to the unusual acidity of the tumor extracellularmicroenvironment (pH_(e)≈6.5), several pH_(e)-responsive nanosystemshave been reported to increase the sensitivity of tumor imaging or theefficacy of therapy. However, for polymer micelle compositions thatrelease drug by hydrolysis in acidic environments, it can take days forthe release of the drug. In that time period, the body can excrete orbreak down the micelles.

To target the acidic endo-/lysosomal compartments, nanovectors withpH-cleavable linkers have been investigated to improve payloadbioavailability. Furthermore, several smart nanovectors with pH-inducedcharge conversion have been designed to increase drug efficacy. Despitethese advances, specific transport and activation of nanoparticles andtheir interactions with different endocytic organelles duringendocytosis in living cells is not well understood. The endocytic systemis comprised of a series of compartments that have distinctive roles inthe sorting, processing and degradation of internalized cargo. Selectivetargeting of different endocytic compartments by pH-sensitivenanoparticles is particularly challenging due to the short nanoparticleresidence times (<mins) and small pH differences in these compartments(e.g. <1 pH unit between early endosomes and lysosomes).

Angiogenesis, the formation of new blood vessels, plays an essentialrole in normal physiological processes such as development and woundrepair. Pathological angiogenesis occurs in tumors as well as a range ofnon-neoplastic diseases (e.g. diabetic retinopathy, endometriosis). Incancer, the formation of new blood vessels from an existing vasculaturenetwork is necessary for sustained tumor growth and exchange ofnutrients and metabolic wastes. In the tumor microenvironment model ofcarcinogenesis, angiogenesis represents the last critical step toovercome the ischemia barrier. Acquisition of the angiogenic phenotyperesults in rapid tumor expansion, as well as facilitation of localinvasion and cancer metastasis.

Tumor angiogenesis is a complex biological process that is orchestratedby a range of angiogenic factors. Initially, stressed tumor cells (e.g.under hypoxia) secrete growth factors and chemokines (e.g. VEGF-A) thatstimulate quiescent vascular endothelium from adjacent host vessels tosprout new capillaries. These growth factors activate or upregulate theexpression of integrins (e.g. α_(v)β₃, □ α_(v)β₅) on blood vessels,which promote endothelial cell migration and survival in the creation ofnew vessel sprouts. Mechanistic understanding of tumor angiogenesis haspropelled the rapid development of a variety of antiangiogenesis agents.Bevacizumab (Avastin®, Genentech) is a humanized anti-VEGF antibody thatinhibits VEGF binding to and signaling through VEGFR1 and VEGFR2receptors that are over-expressed on angiogenic endothelial cells. It isclinically approved in combination with cytotoxic chemotherapy for thetreatment of colorectal cancer, non-small cell lung cancer, and breastcancer. Sunitinib (Sutent®, Pfizer) and sorafenib (Nexavar®, BayerPharm. Corp.) are small molecule inhibitors of multiple receptortyrosine kinases including the VEGF receptors. They have been approvedby the FDA for the treatment of renal cell carcinoma, GI stromal tumors(sunitinib), and unresectable liver cancer (sorafenib). A variety ofother targeted agents are currently in late stage clinical trials (e.g.Vitaxin and Cilengitide, which target α_(v)β₃ integrin, are in phaseII/III clinical trials for treatment of metastatic melanoma and prostatecancer).

Angiogenesis imaging holds considerable promise for early detection ofcancer, as well as post-therapy assessment of many newmolecular-targeted antiangiogenic therapies. Two main strategies,functional and targeted imaging, are currently employed in angiogenesisimaging. Functional imaging strategy measures the blood flow, tumorblood volume and vascular permeability of solid tumors. These imagingtechniques include Doppler ultrasound, dynamic contrast-enhanced CT orMRI. The major advantages are that they can be easily adapted and havealready been clinically implemented to monitor the efficacy ofantiangiogenic drugs. The major drawback is that these methods are notvery specific toward tumor angiogenesis. Recently, targeted imagingstrategy is under intensive investigation with potential advantage ofmore precise characterization of the state of endothelium in a tumor.Among key angiogenesis targets are VEGF and its receptors, integrins(e.g. α_(v)β₃ and α_(v)β₅), and matrix metalloproteases. Various imagingmodalities, such as PET, MRI, optical imaging, ultrasound, are beinginvestigated with different degrees of success.

For cancer molecular imaging applications, achieving high contrastsensitivity and specificity remains a formidable challenge. Currently,most conventional imaging probes utilize an always ON design of contrastprobes and the contrast sensitivity arises from the difference in tissueaccumulation of the imaging payloads. Low tissue concentrations ofintended biomarkers, lack of an amplification strategy to increasesignal output, and high background signals are several major limitingfactors. For small molecular radiotracers (e.g. ⁶⁴Cu-labeled cRGD),although the detection sensitivity is very high (e.g. <10⁻¹² M), thecontrast sensitivity is limited by their relatively low binding affinityto the targeted receptors and insufficient accumulation of imagingpayloads in the targeted tissues. Monoclonal antibodies (mAbs) haveshown superb affinity and specificity to a variety of cancer cellsurface markers. However, radiolabeled or fluorescently labeled mAbs arelimited in molecular imaging applications due to their slow clearancetimes and persistent high background signals in blood. In manyconventional contrast agents, the contrast sensitivity is intrinsicallylimited by the relatively low tissue concentrations of cancer biomarkerson one hand, and high non-specific background signals from the always ONnanoprobes on the other.

What is needed are improved pH-responsive micelle compositions fortherapeutic and diagnostic applications, in particular compositionshaving one or more of: increased imaging and/or drug payloads, prolongedblood circulation times, high contrast sensitivity and specificity,rapid delivery of drug at the target site, and responsiveness withinspecific narrow pH ranges (e.g. for targeting of tumors or specificorganelles).

SUMMARY OF THE INVENTION

In one aspect of the invention is a block copolymer comprising ahydrophilic polymer segment and a hydrophobic polymer segment, whereinthe hydrophilic polymer segment comprises a polymer selected from thegroup consisting of: poly(ethylene oxide) (PEO), poly(methacrylatephosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP), wherein thehydrophobic polymer segment comprises

wherein R′ is —H or —CH₃, wherein R is —NR¹R², wherein R¹ and R² arealkyl groups, wherein R¹ and R² are the same or different, wherein R¹and R² together have from 5 to 16 carbons, wherein R¹ and R² mayoptionally join to form a ring, wherein n is 1 to about 10, wherein x isabout 20 to about 200 in total, and wherein the block copolymeroptionally comprises a labeling moiety. In some embodiments, thehydrophilic polymer segment comprises PEO. In some embodiments, n is 1to 4. In some embodiments, n is 2. In some embodiments, R′ is —CH₃. Insome embodiments, R′ is —H. In some embodiments, x is about 40 to about100 in total. In some embodiments, x is about 50 to about 100 in total.In some embodiments, x is about 40 to about 70 in total. In someembodiments, x is about 60 to about 80 in total. In some embodiments, xis about 70 in total. In some embodiments, R¹ and R² are each straightor branched alkyl. In some embodiments, R¹ and R² join to form a ring.In some embodiments, R¹ and R² are the same. In some embodiments, R¹ andR² are different. In some embodiments, R¹ and R² each have 3 to 8carbons. In some embodiments, R¹ and R² together form a ring having 5 to10 carbons. In some embodiments, R¹ and R² are propyl. In someembodiments, propyl is iso-propyl. In some embodiments, R¹ and R² arebutyl. In some embodiments, butyl is n-butyl. In some embodiments, R¹and R² together are —(CH₂)₅—. In some embodiments, R¹ and R² togetherare —(CH₂)₆—. In some embodiments, the block copolymer comprises acompound of Formula I:

wherein L is a labeling moiety, wherein y is 0 to about 6, wherein R″ is—H or —CH₃; wherein m is 1 to about 10; wherein z is such that the PEOis about 2 kD to about 20 kD in size, wherein R′″ is any suitablemoiety, and wherein the following portion of the structure:

may be arranged in any order. In some embodiments, R″ is —CH₃. In someembodiments, R″ is —H. In some embodiments, m is 1 to 4. In someembodiments, m is 2. In some embodiments, the PEO is about 2 kD to about10 kD in size. In some embodiments, the PEO is about 4 kD to about 6 kDin size. In some embodiments, the PEO is about 5 kD in size. In someembodiments, z is about 114. In some embodiments, y is 0. In someembodiments, y is 1 to 6. In some embodiments, y is about 3. In someembodiments, L is a fluorescent label. In some embodiments, thefluorescent label is tetramethyl rhodamine (TMR). In some embodiments, Lis a near-infrared (NIR) label. In some embodiments, the NIR label iscypate. In some embodiments, the NIR label is a cypate analog. In someembodiments, R′″ is an end group resulting from a polymerizationreaction. In some embodiments, R′″ is Br. In some embodiments, R′″ isthiolate. In some embodiments, R′ is a thioester. In some embodiments,the following portion of the structure:

is randomized. In some embodiments, the block copolymer forms apH-sensitive micelle.

In another aspect of the invention is a composition comprising apH-sensitive micelle, wherein the pH-sensitive micelle comprises a blockcopolymer as described herein. It is to be understood that any of theblock copolymers described herein may be utilized in making apH-sensitive micelle. In some embodiments, the micelle has a size ofabout 10 to about 200 nm. In some embodiments, the micelle has a size ofabout 20 to about 100 nm. In some embodiments, the micelle has a size ofabout 30 to about 50 nm. In some embodiments, the micelle has a pHtransition range of less than about 1 pH unit. In some embodiments, themicelle has a pH transition range of less than about 0.5 pH unit. Insome embodiments, the micelle has a pH transition range of less thanabout 0.25 pH unit. In some embodiments, the micelle has a pH transitionvalue of about 5 to about 8. In some embodiments, the micelle has a pHtransition value of about 5 to about 6. In some embodiments, the micellehas a pH transition value of about 6 to about 7. In some embodiments,the micelle has a pH transition value of about 7 to about 8. In someembodiments, the micelle has a pH transition value of about 6.3 to about6.9. In some embodiments, the micelle has a pH transition value of about5.0 to about 6.2. In some embodiments, the micelle has a pH transitionvalue of about 5.9 to about 6.2. In some embodiments, the micelle has apH transition value of about 5.0 to about 5.5. In some embodiments, themicelle further comprises a targeting moiety. In some embodiments, thetargeting moiety binds to VEGFR2. In some embodiments, the targetingmoiety is a Fab′ fragment of RAFL-1 mAb. In some embodiments, thetargeting moiety binds to α_(v)β₃ integrin. In some embodiments, thetargeting moiety is cRGDfK. In some embodiments, the targeting moietybinds to an angiogenesis biomarker. In some embodiments, theangiogenesis biomarker is VEGF-VEGFR complex or endoglin. In someembodiments, the composition further comprises a drug encapsulatedwithin the micelle. In some embodiments, the drug is hydrophobic. Insome embodiments, the drug has a log p of about 2 to about 8. In someembodiments, the drug is a chemotherapeutic agent. In some embodiments,the drug is doxorubicin. In some embodiments, the drug isbeta-lapachone. In some embodiments, the drug is paclitaxel.

In another aspect of the invention is a method for treating cancer in anindividual in need thereof, comprising administration of an effectiveamount of a pH-sensitive micelle composition comprising achemotherapeutic agent as described herein. In some embodiments, thecancer comprises a solid tumor.

In another aspect of the invention is a method for imaging a tumor in anindividual, comprising a) administering a pH-sensitive micellecomposition as described herein to the individual, wherein the blockcopolymer comprises a labeling moiety, and b) determining thedistribution of the block copolymer in its disassociated form. In someembodiments, the method is used to diagnose a tumor in the individual.In some embodiments, the method is used to monitor a tumor in theindividual.

In another aspect of the invention is a method for delivery of a drug toearly endosomes, comprising administration of a pH-sensitive micellecomposition comprising a drug as described herein to an individual inneed thereof, wherein the micelle has a pH transition value of about 5.9to about 6.5.

In another aspect of the invention is a method for delivery of a drug tolate endosomes or lysosomes, comprising administration of a pH-sensitivemicelle composition comprising a drug as described herein to anindividual in need thereof, wherein the micelle has a pH transitionvalue of about 5.0 to about 5.5. In some embodiments, the drug is alysosomal storage disease drug.

In another aspect of the invention is a method for imaging earlyendosomal activity in an individual, comprising a) administration of apH sensitive micelle composition as described herein to the individual,wherein the block copolymer comprises a labeling moiety, and wherein themicelle has a pH transition value of about 5.9 to about 6.5, and b)determining the distribution of the block copolymer in its disassociatedform.

In another aspect of the invention is a method for imaging lateendosomal or lysosomal activity in an individual, comprising a)administration of a pH sensitive micelle composition as described hereinto the individual, wherein the block copolymer comprises a labelingmoiety, and wherein the micelle has a pH transition value of about 5.0to about 5.5, and b) determining the distribution of the block copolymerin its disassociated form.

In another aspect of the invention is a compound of the formula:

In another aspect of the invention is a polymer of the compound C6A-MA.

In another aspect of the invention is a compound of the formula:

In another aspect of the invention is a polymer of the compound C7A-MA.

In another aspect of the invention is a compound of the formula:

In another aspect of the invention is a polymer of the compound DBA-MA.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate examples of block copolymers of theinvention.

FIG. 1C illustrates the design principle of an example of a micellecomprising a fluorescent label (using TMR as an example). At high pH,micelle assembly results in fluorescence quenching due to homoFRET andphotoinduced electron transfer (PET) mechanisms. At low pH, micelledisassembly leads to dramatic increase in emission. At high pH, theamine in the hydrophobic polymer segment is not protonated. At low pH,the amine group in the hydrophobic polymer segment is protonated.

FIG. 2A illustrates an example of synthesis PEO-b-PR copolymers by atomtransfer radical polymerization (ATRP) method.

FIG. 2B illustrates an example of synthesis of PEO-b-(PR-r-TMR)nanoprobes.

FIG. 3A shows the normalized fluorescence intensity of pHAM nanoprobes3, 4, 6, 7 as a function of pH. The pH response (ΔpH_(10-90%)) was <0.25pH unit and F_(max)/F_(min) was up to 55 fold.

FIG. 3B shows stopped-flow fluorescence measurement of nanoprobe 4(pH_(t)=5.4) after pH activation at 4.9. Fluroesence recovery time(τ_(1/2)) was 3.7 ms.

FIG. 4A shows the pH titration curves of two representative PEO-b-PRblock copolymers, 5 and 7, and their corresponding monomers.

FIG. 4B shows deuterated ¹H NMR spectra of two representative PEO-b-PRblock copolymers, 5 and 7, at different ionization states of tertiaryamines.

FIG. 4C shows transmission electron microscopy (TEM) of PEO-b-PR blockcopolymer 7 in aqueous solution, demonstrating the formation of micellesabove its pKa (6.7) at pH 7.4 and complete micelle dissociation at pH5.5. Average diameter of micelles was 45 nm.

FIGS. 5A and 5B show quantification of activation of pHAM nanoparticlesin H2009 cells and culture medium upon acidification. FIG. 5A showssignal to noise ratios (SNRs) of 3 inside H2009 cells and medium overtime. FIG. 5B shows a comparison of SNR between H2009 cells and mediumbefore and after the addition of HCl. A large contrast(SNR_(Cell)/SNR_(Med)=31 at 60 min) was observed before HCl addition andthe trend is reversed (SNR_(Cell)/SNR_(Med)=0.74) after HCl. P-valueswere calculated using the Student's t-test.

FIG. 6A shows an examination of the subcellular locations (earlyendosomes (Rab5a) and late endosomes/lysosomes (Lamp1)) for pHAMactivation of nanoprobe 3 over time using confocal imaging.

FIG. 6B shows an examination of the subcellular locations (earlyendosomes (Rab5a) and late endosomes/lysosomes (Lamp1)) for pHAMactivation of nanoprobe 4 over time using confocal imaging.

FIG. 6C and FIG. 6D depict the different processes of intracellularuptake and activation of the two nanoprobes.

FIG. 7 shows doxorubicin release from PEO-b-PC6A micelles at differenttime points in various pH environments.

FIG. 8 illustrates syntheses of NIR-NHS ester and PEO-b-(PR-r-NIR)copolymers for the development of NIR-pHAM.

FIG. 9 illustrates syntheses of maleimide-terminated PEG-b-PRcopolymers.

FIG. 10A shows fluorescence intensity of HUVEC cells differently treatedwith cRGD-encoded pHAM nanoprobes, cRAD-pHAM, free cRGD block (N>10 foreach group) and cell culture medium, respectively.

FIG. 10B shows contrast to noise ratio (CNR) of HUVEC cells treated withcRGD-pHAM over the cRAD-pHAM and dRGD block controls.

FIG. 11 shows the in vivo pharmacokinetics studies of cRGD-encoded pHAM(targeted micelles) and cRGD-free pHAM (nontargeted micelles) in A549tumor-bearing mice.

FIG. 12 illustrates an example of pH-activatable (pHAM) nanoprobes forimaging of angiogenesis biomarkers (e.g. VEGFR2, α_(v)β₃) invascularized tumors. These nanoprobes will stay “silent” (or OFF state)during blood circulation, but can be turned ON by pH activation afterreceptor-mediated endocytosis in angiogenic tumor endothelial cells.

FIG. 13 illustrates an example of intracellular activation mechanism fora vascular targeted pHAM inside acidic intracellular organelles (i.e.endosomes/lysosomes).

FIGS. 14A and 14B show pH-dependent micellization behaviors ((14A)normalized light scattering intensity and (14B) pyrene I₁/I₃ emissionratio as a function of pH) from 4 different PEG-b-PR copolymers having aconcentration at 0.1 mg/ml.

FIG. 15 illustrates selective targeting of drug delivery to a tumor by alarger macromolecule such as a micelle of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides block copolymers and micelle compositionscomprising one or more of said block copolymers that are useful in oneor more therapeutic and/or diagnostic applications, such as treatment ofcancer, cardiovascular disease, inflammation, an autophagy-relateddisease, or lysosomal storage disease, tumor imaging, and/or imaging ofintracellular organelles such as early endosomes, late endosomes andlysosomes. The invention further provides methods for using the micellecompositions in such therapeutic and diagnostic applications.

The block copolymers of the invention comprise a hydrophilic polymersegment and a hydrophobic polymer segment, wherein the hydrophobicpolymer segment comprises an ionizable amine group to render pHsensitivity. The block copolymers form pH-activatable micellar (pHAM)nanoparticles based on the supramolecular self-assembly of theseionizable block copolymers (see e.g. FIG. 1C). For example, FIG. 1Cillustrates the design principle of a non-limiting example of a micelleof the invention. At higher pH, the block copolymers assemble intomicelles, whereas at lower pH, ionization of the amine group in thehydrophobic polymer segment results in dissociation of the micelle.Without wishing to be bound by theory, micelle formation and itsthermodynamic stability are driven by the delicate balance between thehydrophobic and hydrophilic segments. The ionizable groups may act astunable hydrophilic/hydrophobic blocks at different pH values, which maydirectly affect the dynamic self-assembly of micelles. Without wishingto be bound by theory, micellization may sharpen the ionizationtransition of the amines in the hydrophobic polymer segment, renderingfast and ultra-sensitive pH response. Different block copolymers may beselected to provide micelles having different transition pH valueswithin physiological range, in order to achieve selective activationwithin various environments, such as tumors (e.g. the extracellularenvironment of tumors), or within specific endocytic compartments suchas early or late endosomes or lysosomes.

For therapeutic applications, a drug may be incorporated into theinterior of the micelles. Specific pH conditions (e.g. acidic pH presentin tumors and endocytic compartments) may lead to rapid protonation anddissociation of micelles into unimers, thereby releasing the drug. Insome embodiments, the micelle provides stable drug encapsulation atphysiological pH (pH 7.4), but can quickly release the drug in acidicenvironments. The micelles of the invention may provide one or moreadvantages in therapeutic applications, such as: (1) disassociation ofthe micelle (and rapid release of drug) within a short amount of time(e.g. within minutes) under certain pH environments (e.g. acidicenvironments), as opposed to hours or days for previous micellecompositions; (2) encapsulation of a high percentage of drug; (3)selective targeting of drug delivery to the desired site (e.g. tumor orlysosome), which may enhance drug efficacy and reduce toxicity tohealthy cells (see e.g. FIG. 15); (4) prolonged blood circulation times;(5) responsiveness within specific narrow pH ranges (e.g. for targetingof specific organelles), and (6) image-guided therapy, where imagingsignals can be a predictive factor for the therapeutic efficacy for thetreatment.

For diagnostic applications, a labeling moiety may be conjugated to theblock copolymer. In some embodiments, the label (e.g. a fluorescentlabel) is sequestered inside the micelle when the pH favors micelleformation, and sequestration results in a decrease in label signal (e.g.via fluorescence quenching, see e.g. FIG. 1C). Specific pH conditions(e.g. acidic pH present in tumors and endocytic compartments) may leadto rapid protonation and dissociation of micelles into unimers, therebyexposing the label, and increasing the label signal (e.g. increasingfluorescence emission). The micelles of the invention may provide one ormore advantages in diagnostic applications, such as: (1) disassociationof the micelle (and rapid increase in label signal) within a shortamount of time (e.g. within minutes) under certain pH environments (e.g.acidic environments), as opposed to hours or days for previous micellecompositions; (2) increased imaging payloads; (3) selective targeting oflabel to the desired site (e.g. tumor or particular endocyticcompartment); (4) prolonged blood circulation times; (5) responsivenesswithin specific narrow pH ranges (e.g. for targeting of specificorganelles); and (6) high contrast sensitivity and specificity. Forexample, the micelles may stay silent (or in the OFF state) with minimumbackground signals under normal physiological conditions (e.g. bloodcirculation), but imaging signals can be greatly amplified when themicelles reach their intended molecular targets in vivo (e.g.extracellular tumor environment or cellular organelle). As anon-limiting example, upon specific targeting to angiogenic biomarkers(e.g. α_(v)β₃), micelle nanoprobes can be turned ON by pH activationinside endosomes/lysosomes after receptor-mediated endocytosis. FIG. 12illustrates an example of pH-activatable (pHAM) nanoprobes for imagingof angiogenesis biomarkers (e.g. VEGFR2, α_(v)β₃) in vascularizedtumors. These nanoprobes will stay “silent” (or OFF state) during bloodcirculation, but can be turned ON by pH activation afterreceptor-mediated endocytosis in angiogenic tumor endothelial cells.FIG. 13 illustrates the intracellular activation mechanism for avascular targeted pHAM inside acidic intracellular organelles (i.e.endosomes/lysosomes).

Definitions

As used herein, “alkyl” indicates any saturated hydrocarbon moiety,including, for example, straight chain, branched chain, or cyclic(including fused and spiro bicyclic and polycyclic) saturatedhydrocarbon moieties which may optionally be substituted with one ormore additional saturated hydrocarbon moieties.

As used herein, “pH-sensitive micelle”, “pH-activatable micelle” and“pH-activatable micellar (pHAM) nanoparticle” are used interchangeablyherein to indicate a micelle comprising one or more block copolymers,which disassociates depending on the pH (e.g. above or below a certainpH). As a non-limiting example, at a certain pH, the block copolymer issubstantially in micellar form. As the pH changes (e.g. decreases), themicelles begin to disassociate, and as the pH further changes (e.g.further decreases), the block copolymer is present substantially indisassociated (non-micellar) form.

A “nanoprobe” is used herein to indicate a pH-sensitive micelle whichcomprises an imaging labeling moiety.

As used herein, “pH transition range” indicates the pH range over whichthe micelles disassociate. In some embodiments, the pH transition rangeis the pH response sharpness. An example of determining pH responsesharpness is described in Example 2 below. Briefly, the fluorescenceintensity versus pH is measured for a block copolymer which comprises afluorescent label that is sequestered within the micelle (quenchingfluorescence) when the block copolymer is in micellar form (see e.g.FIG. 1C). As the pH changes (e.g. decreases), the micelle disassociates,exposing the fluorescent label and resulting in fluorescence emission.Normalized fluorescence intensity (NFI) vs. pH curves permitquantitative assessment of the pH responsive properties of the micelle.NFI is calculated as the ratio of [F−F_(min)]/[F_(max)−F_(min)], where Fis the fluorescence intensity of the micelle at any given pH, andF_(max) and F_(min) are the maximal and minimal fluorescence intensitiesat the ON/OFF states, respectively. pH response sharpness isΔpH_(10-90%), the pH range in which the NFI value varies from 10% to90%. For label-free copolymers, dynamic light scattering (DLS) or anexternal fluorophore (e.g. pyrene) can be used to characterize thepH-dependent micellization behaviors. For example, FIG. 14A shows thenormalized light scattering intensity of several PEO-b-PR copolymers at0.1 mg/mL concentration as a function of pH. At different pH values,dramatic increase of light scattering intensity was observed due to theformation of micelle nanoparticles from unimers in solution. Thehydrodynamic diameters of the resulting micelles were measured at 40-50nm. The light scattering data was further supported by examining theI₁/I₃ ratios (at 372-374 and 382-384 nm, respectively) of pyreneemissions (λ_(ex)=339 nm) (FIG. 14B). I₁/I₃ ratio reflects the polarityof the pyrene environment where a partition of pyrene in the micellecore leads to decreased I₁/I₃ values.

As used herein, “pH transition value” (pH_(t)) indicates the pH at whichhalf of the micelles are disassociated. An example of determining pHtransition value is described in Example 2 below. Briefly, for a blockcopolymer which comprises a fluorescent label that is sequestered withinthe micelle (quenching fluorescence) when the block copolymer is inmicellar form, the pH transition value is the pH at which thefluorescence emission measured is 0.5×(F_(max)+F_(min)), where F_(max)and F_(min) are the maximal and minimal fluorescence intensities at theON/OFF states, respectively. For label-free copolymers, dynamic lightscattering (DLS) or an external fluorophore (e.g. pyrene) can be used tocharacterize the pH-dependent micellization behaviors. For example, FIG.14A shows the normalized light scattering intensity of several PEO-b-PRcopolymers at 0.1 mg/mL concentration as a function of pH. At differentpH values, dramatic increase of light scattering intensity was observeddue to the formation of micelle nanoparticles from unimers in solution.The hydrodynamic diameters of the resulting micelles were measured at40-50 nm. The light scattering data was further supported by examiningthe I₁/I₃ ratios (at 372-374 and 382-384 nm, respectively) of pyreneemissions (λ_(ex)=339 nm) (FIG. 14B). I₁/I₃ ratio reflects the polarityof the pyrene environment where a partition of pyrene in the micellecore leads to decreased I₁/I₃ values. Both light scattering and pyreneexperiments yielded similar pH transition values. The pH_(t) values were5.0, 6.2, 7.0, and 7.2 for PEO-b-PDBA, PEO-b-PDPA, PEO-b-PC7A,PEO-b-PC6A, respectively.

As used herein, the term “treating” refers to a clinical interventiondesigned to alter the natural course of clinical pathology of thedisease or disorder being treated (e.g., cancer). Desirable effects oftreatment include, for example, ameliorating or palliating the diseasestate, slowing or reversing the progression of the disorder, remission,or improved prognosis.

As used herein, the term “effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic, prophylactic, or diagnostic result. An effectiveamount can be provided in one or more administrations.

As used herein, “individual” indicates an animal, preferably a mammal,including humans, primates, laboratory animals (e.g. rats, mice, etc.),farm animals (e.g. cows, sheep, goats, pigs, etc.), pets (e.g. dogs,cats, etc.), and sport animals (e.g. horses, etc.). In some embodiments,an individual is a human.

Reference to “about” a value or parameter herein also includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

It is understood that all aspects and embodiments of the inventiondescribed herein include “comprising,” “consisting,” and “consistingessentially of” aspects and embodiments. It is to be understood thatmethods or compositions “consisting essentially of” the recited elementsinclude only the specified steps or materials and those that do notmaterially affect the basic and novel characteristics of those methodsand compositions.

It is to be understood that any of the compositions described herein maybe used in any of the methods as described herein, unless contextclearly indicates otherwise.

Block Co-Polymer Compounds

Novel block copolymers are provided herein, comprising a hydrophilicpolymer segment and a hydrophobic polymer segment, wherein thehydrophilic polymer segment comprises a polymer selected from the groupconsisting of: poly(ethylene oxide) (PEO), poly(methacrylatephosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP), wherein thehydrophobic polymer segment comprises

wherein R′ is —H or —CH₃, wherein R is —NR¹R², wherein R¹ and R² arealkyl groups, wherein R¹ and R² are the same or different, wherein R¹and R² together have from 5 to 16 carbons, wherein R¹ and R² mayoptionally join to form a ring, wherein n is 1 to about 10, wherein x isabout 20 to about 200 in total, and wherein the block copolymer mayfurther optionally comprise a labeling moiety. For example, x may beabout 20 to about 200 as a continuous segment (i.e. a continuous segmentof about 20 to about 200 monomer units), or other moieties (e.g.moieties comprising a label) may be interspersed between the monomerunits, for example as described in more detail below.

Block copolymers of the invention include, for example, compounds ofFormula I:

wherein L is a labeling moiety, wherein y is 0 to about 6, wherein R″ is—H or —CH₃; wherein m is 1 to about 10, wherein z is such that the PEOis about 2 kD to about 20 kD in size, wherein x, n, R, and R′ are asdefined above, wherein R′″ is any suitable moiety, and wherein thefollowing portion of the structure:

may be arranged in any order.

In some embodiments, R′″ is an end group resulting from a polymerizationreaction. For example, R′″ may be —Br when atom transfer radicalpolymerization (ATRP) is used. It is to be understood that the chemicalstructures in FIGS. 1A, 2A, 2B, 8 and 9 may comprise a —Br as the endgroup resulting from the polymerization reaction. For example, R′″ maybe a sulfur-containing group such as thiolate or a thioester whenreversible addition-fragmentation chain transfer (RAFT) is used. In someembodiments, R′″ is —Br. In some embodiments, R′″ is thiolate. In someembodiments, R′″ is a thioester. The end group may optionally be furthermodified following polymerization with an appropriate moiety.

In some embodiments, the following portion of the structure:

is randomized, i.e.:

wherein r indicates a random ordering of the R containing moieties andthe L containing moieties (i.e. the R containing moieties and the Lcontaining moieties are randomly interspersed).

In some embodiments, the following portion of the structure:

is arranged sequentially. For example, the R containing moieties may bepresent as a single block, with the L containing moieties present as asingle block either preceding or following the R containing moieties.Other arrangements may also be utilized.

Hydrophilic Polymer Segment

In some embodiments, the hydrophilic polymer segment comprisespoly(ethylene oxide) (PEO). In some embodiments, the hydrophilic polymersegment comprises poly(methacrylate phosphatidyl choline) (MPC). In someembodiments, the hydrophilic polymer segment comprisespolyvinylpyrrolidone (PVP). In general, the PEO, MPC, or PVP polymer inthe hydrophilic polymer segment is about 2 kD to about 20 kD in size. Insome embodiments, the polymer is about 2 kD to about 10 kD in size. Insome embodiments, the polymer is about 2 kD to about 5 kD in size. Insome embodiments, the polymer is about 3 kD to about 8 kD in size. Insome embodiments, the polymer is about 4 kD to about 6 kD in size. Insome embodiments, the polymer is about 5 kD in size. In someembodiments, the polymer has about 100 to about 130 monomer units. Insome embodiments, the polymer has about 110 to about 120 monomer units.In some embodiments, the polymer has about 114 monomer units. In someembodiments, the polydispersity index (PDI) of the polymer is less thanabout 1.2. In some embodiments, the polydispersity index (PDI) of thepolymer is less than about 1.1.

Suitable PEO, MPC, and PVP polymers may be purchased (for example, PEOpolymers may be purchased from Aldrich Sigma) or may be synthesizedaccording to methods known in the art. In some embodiments, thehydrophilic polymer can be used as an initiator for polymerization ofthe hydrophobic monomers to form a block copolymer.

For example, MPC polymers (e.g. narrowly distributed MPC polymers) canbe prepared by atom transfer radical polymerization (ATRP) withcommercially available small molecule initiators such as ethyl2-bromo-2-methylpropanoate (Sigma Aldrich). These resulting MPC polymerscan be used as macromolecular ATRP initiators to further copolymerizewith other monomers to form block polymers such as MPC-b-PDPA. PEO-b-PRblock copolymers can be synthesized using atom transfer radicalpolymerization (ATRP) or reversible addition-fragmentation chaintransfer (RAFT) methods (See e.g. Australian Journal of ChemistryVolume: 58 Issue: 6 Pages: 379-410 (2005); Progress in Polymer ScienceVolume: 32 Issue: 1 Pages: 93-146 (2007). ATRP or RAFT allows for livingpolymerization which can yield PEO-b-PR copolymers with narrowpolydispersity (<1.1). Different metharylate or acrylate monomers can beused to produce PR segments with different pH sensitivity.

Hydrophobic Polymer Segment

The hydrophobic polymer segment comprises:

wherein R′ is —H or —CH₃, wherein R is —NR¹R², wherein R¹ and R² arealkyl groups, wherein R¹ and R² are the same or different, wherein R¹and R² together have from 5 to 16 carbons, wherein R¹ and R² mayoptionally join to form a ring, wherein n is 1 to about 10, and whereinx is about 20 to about 200 in total.

In some embodiments, n is 1 to 4. In some embodiments, n is 2. Invarious embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, R′ is —CH₃. In some embodiments, R′ is —H.

In some embodiments, x is about 40 to about 100 in total. In someembodiments, x is about 50 to about 100 in total. In some embodiments, xis about 40 to about 70 in total. In some embodiments, x is about 60 toabout 80 in total. In some embodiments, wherein x is about 70 in total.

In some embodiments, R¹ and R² together have from 5 to 14 carbons. Insome embodiments, R¹ and R² together have from 5 to 12 carbons. In someembodiments, R¹ and R² together have from 5 to 10 carbons. In someembodiments, R¹ and R² together have from 5 to 8 carbons. In someembodiments, R¹ and R² together have from 6 to 12 carbons. In someembodiments, R¹ and R² together have from 6 to 10 carbons. In someembodiments, R¹ and R² together have from 6 to 8 carbons. In variousembodiments, R¹ and R² together have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 carbons. In some embodiments, R¹ and R² each have 3 to 8carbons. In some embodiments, R¹ and/or R² comprise 3 carbons. In someembodiments, R¹ and/or R² comprise 4 carbons. In some embodiments, R¹and/or R² comprise 5 carbons. In some embodiments, R¹ and/or R² comprise6 carbons. In some embodiments, R¹ and/or R² comprise 7 carbons. In someembodiments, R¹ and/or R² comprise 8 carbons. In some embodiments, R¹and R² are the same. In some embodiments, R¹ and R² are different. Insome embodiments, R¹ and R² are each independently straight or branchedalkyl. In some embodiments, R¹ and R² are each straight alkyl. In someembodiments, R¹ and R² are each branched alkyl. Suitable alkyl groupsfor R¹ and R² include, for example, methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, and pentadecyl, including various possible skeletal isomersfor each alkyl group such as n-, iso-, sec-, tert-, neo-, etc., providedthe total number of carbons in R is from 5 to 16. In some embodiments,R¹ and R² are propyl. In some embodiments, propyl is iso-propyl. In someembodiments, propyl is n-propyl. In some embodiments, R¹ and R² arebutyl. In some embodiments, butyl is n-butyl. In some embodiments, butylis iso-butyl. In some embodiments, butyl is sec-butyl. In someembodiments, butyl is t-butyl. In some embodiments, R¹ and R² join toform a ring. The ring may optionally be substituted with one or morealkyl groups, provided the total number of carbons in R is from 5 to 16.In some embodiments, R¹ and R² together form a ring having 5 to 10carbons. In some embodiments, R¹ and R² together form a ring having 5 to8 carbons. In some embodiments, R¹ and R² together form a ring having 5to 7 carbons. In some embodiments, R¹ and R² together are —(CH₂)₅—. Insome embodiments, R¹ and R² together are —(CH₂)₆—.

The hydrophobic polymer segment may be synthesized according to, e.g.Atom Transfer Radical Polymerization (ATRP) or reversibleaddition-fragmentation chain transfer (RAFT). An example of ATRPsynthesis of a hydrophobic polymer segment may be found in Example 1. Insome embodiments, the polydispersity index (PDI) for the hydrophobicpolymer segment is less than about 1.2. In some embodiments, thepolydispersity index (PDI) for the hydrophobic polymer segment is lessthan about 1.1.

Labeling Moiety

The block copolymer may optionally comprise one or more labelingmoieties (e.g. 1, 2, 3, 4, 5, 6, or more). In some embodiments, thelabel is a fluorescent label. In some embodiments, the fluorescent labelis tetramethyl rhodamine (TMR). In some embodiments, the label is anear-infrared (NIR) label. In some embodiments, the NIR label is cypateor a cypate analog.

When the block copolymer is a compound of Formula I, in someembodiments, R″ is —CH₃. In some embodiments, R″ is —H. In someembodiments, m is 1 to 4. In some embodiments, m is 2. In variousembodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments,y is 0. In some embodiments, y is 1 to 6. In various embodiments, y is1, 2, 3, 4, 5, or 6. In some embodiments, y is 3.

The labeling moiety may be conjugated to the copolymer directly orthrough a linker moiety. Methods known in the art may be used toconjugate the labeling moiety to, for example, the hydrophobic polymer.Examples of conjugation may be found in, for example, Examples 1 and 5below.

The micelles of the invention may advantageously have high imagingpayloads. In various embodiments, the micelles have at least about 500dyes, at least about 1000, at least about 1500, at least about 2000, atleast about 2400, at least about 3000 dyes per micelle. In comparison,typical immunofluorescent conjugates have 4 fluorophores per molecule,as a higher number will lead to dye quenching and may also modifybinding epitopes.

Different labels may be preferred for the particular method of use. Forexample, tetramethylrhodamine may be used, e.g., for in vitro cell studyon confocal imaging, while for animal imaging studies in vivo, NIR dyesmay increase the tissue penetrations.

Exemplary Block Copolymers

Non-limiting examples of block copolymers of the invention include thosedescribed in the Examples below. Non-limiting examples of blockcopolymers of Formula I are provided in Table A.

TABLE A Exemplary block copolymers

Compound R′ R¹/R² n z R″ m x y L R′′′ 3 (no label) —CH₃ iPr/iPr 2 114 —— 45 0 None Br 4 (no label) —CH₃ nBu/nBu 2 114 — — 51 0 None Br 6 (nolabel) —CH₃ —(CH₂)₅— 2 114 — — 45 0 None Br 7 (no label) —CH₃ —(CH₂)₆— 2114 — — 49 0 None Br 3 (TMR —CH₃ iPr/iPr 2 114 —CH₃ 2 70 3 TMR Br label)4 (TMR —CH₃ nBu/nBu 2 114 —CH₃ 2 70 3 TMR Br label) 6 (TMR —CH₃ —(CH₂)₅—2 114 —CH₃ 2 70 3 TMR Br label) 7 (TMR —CH₃ —(CH₂)₆— 2 114 —CH₃ 2 70 3TMR Br label) 3 (cypate —CH₃ iPr/iPr 2 114 —CH₃ 2 70 3 cypate Br label)4 (cypate —CH₃ nBu/nBu 2 114 —CH₃ 2 70 3 cypate Br label) 6 (cypate —CH₃—(CH₂)₅— 2 114 —CH₃ 2 70 3 cypate Br label) 7 (cypate —CH₃ —(CH₂)₆— 2114 —CH₃ 2 70 3 cypate Br label)

In Table A, the following portion of the structure:

is randomized, i.e.:

With regards to the compounds described herein, it is to be understoodthat polymerization reactions may result in a certain variability ofpolymer length, and that the numbers described herein indicating thenumber of monomer units within a particular polymer (e.g. x, y, z) mayindicate an average number of monomer units. In some embodiments, apolymer segment described herein (e.g. the hydrophobic polymer segment,the hydrophilic polymer segment) has a polydispersity index (PDI) lessthan about 1.2. In some embodiments, the polydispersity index (PDI) forthe polymer segment is less than about 1.1. In some embodiments, thepolydispersity index (PDI) for the block copolymer is less than about1.2. In some embodiments, the polydispersity index (PDI) for the blockcopolymer is less than about 1.1.

Micelle Compositions

One or more block copolymers (e.g. 2, 3, 4, 5, or more) described hereinmay be used to form a pH-sensitive micelle. In some embodiments, acomposition comprises a single type of micelle. In some embodiments, twoor more (e.g. 2, 3, 4, 5, or more) different types of micelles may becombined to form a mixed-micelle composition.

The pH-sensitive micelle compositions of the invention mayadvantageously have a narrow pH transition range, in contrast to otherpH sensitive compositions in which the pH response is very broad (i.e. 2pH units). In some embodiments, the micelles have a pH transition rangeof less than about 1 pH unit. In various embodiments, the micelles havea pH transition range of less than about 0.9, less than about 0.8, lessthan about 0.7, less than about 0.6, less than about 0.5, less thanabout 0.4, less than about 0.3, less than about 0.2, less than about 0.1pH unit. In some embodiments, the micelles have a pH transition range ofless than about 0.5 pH unit. In some embodiments, the micelles have a pHtransition range of less than about 0.25 pH unit.

When the micelles comprise a fluorescent label, the narrow pH responsiveproperties of pHAM may improve the efficiency of fluorescencegeneration. Without wishing to be bound by theory, the pH response ofpHAM may originate from both homoFRET and PET mechanisms as a result ofthe cooperative neutralization and micellization of the block copolymers(see e.g. FIG. 1C). Compared with small molecular pH-sensitive dyes orPET-based micelles (activations need 2 pH units), the sharpened pHresponse from pHAM may result in complete turn-ON of the fluorophoreswith subtle changes of pH in tumor microenvironment (pH_(e)=6.5-6.9) orintracellular organelles (5.0-6.2).

The micelles may have different pH transition values withinphysiological range, in order to target specific cells ormicroenvironments. In some embodiments, the micelles have a pHtransition value of about 5 to about 8. In some embodiments, themicelles have a pH transition value of about 5 to about 6. In someembodiments, the micelles have a pH transition value of about 6 to about7. In some embodiments, the micelles have a pH transition value of about7 to about 8. In some embodiments, the micelles have a pH transitionvalue of about 6.3 to about 6.9 (e.g. tumor microenvironment). In someembodiments, the micelles have a pH transition value of about 5.0 toabout 6.2 (e.g. intracellular organelles). In some embodiments, themicelles have a pH transition value of about 5.9 to about 6.2 (e.g.early endosomes). In some embodiments, the micelles have a pH transitionvalue of about 5.0 to about 5.5 (e.g. late endosomes or lysosomes). Asdescribed in the Examples, nanoprobes 4, 3, 7 and 6 had fluorescencetransition pH values of 5.4, 6.3, 6.8 and 7.2, respectively.

Labeled micelles of the invention may advantageously have a large signalresponse (e.g. a larger difference in signal between ON and OFF states).For example, when fluorescent labels are used, the ratio of F_(max) andF_(min) (R_(F)=F_(max)/F_(min)) can be used to quantify the fluorescenceresponse between the ON/OFF states. As shown in the Examples, nanoprobeshaving R_(F) values in the range of 10 to 55 fold were made (Table 3),demonstrating the large fluorescence response of the nanoprobes. Invarious embodiments, labeled micelles have a signal response of at leastabout 10, at least about 20, at least about 30, at least about 40, atleast about 50, at least about 60.

Without wishing to be bound by theory, the use of micelles in cancertherapy may enhance anti-tumor efficacy and reduce toxicity to healthytissues, in part due to the size of the micelles. While small moleculessuch as certain chemotherapeutic agents (e.g. doxorubicin) can enterboth normal and tumor tissues, non-targeted micelle nanoparticles maypreferentially cross leaky tumor vasculature (see e.g. FIG. 15). In someembodiments, the micelles have a size of about 10 to about 200 nm. Insome embodiments, the micelles have a size of about 20 to about 100 nm.In some embodiments, the micelles have a size of about 30 to about 50nm.

Examples of methods of generating micelles from block copolymers may befound in the Examples below. For example, block copolymer is firstdissolved in organic solvent (e.g. THF) and may be added to an aqueoussolution, optionally under sonication, wherein the copolymerself-assemble to form micelles in the solution.

In some embodiments, the micelle further comprises a drug. In someembodiments, the micelle further comprises a labeling moiety. In someembodiments, the micelle further comprises a targeting moiety. In someembodiments, the micelle further comprises a drug and a labeling moiety.In some embodiments, the micelle further comprises a drug and atargeting moiety. In some embodiments, the micelle further comprises atargeting moiety and a labeling moiety. In some embodiments, the micellefurther comprises a drug, a targeting moiety, and a labeling moiety.

Targeting Moieties

The micelles may further optionally comprise a targeting moiety intherapeutic or diagnostic applications. For example, a targeting moietycan target a cancer cell surface marker, such as an angiogenesisbiomarker. For example, in diagnostic applications, targeted nanoprobesmay be useful for diagnosing tumors and/or the efficacy assessment ofmolecular-targeted antiangiogenic therapies, where the expression levelsof the therapeutic targets (e.g. VEGFR2, □α_(v)β₃) can be specificallymeasured.

In some embodiments, the targeting moiety binds to an angiogenesisbiomarker. In some embodiments, the angiogenesis biomarker is VEGF-VEGFRcomplex or endoglin. In some embodiments, the targeting moiety binds toVEGFR2. In some embodiments, the targeting moiety is a Fab′ fragment ofRAFL-1 mAb. In some embodiments, the targeting moiety binds to α_(v)β₃integrin. In some embodiments, the targeting moiety is cRGDfK.

The targeting moiety may be conjugated to the block copolymer (e.g., thehydrophilic polymer segment) by methods known in the art. Examples ofconjugation may be found in the Examples below.

Drug Encapsulation

The micelles may further optionally comprise a drug encapsulated withinthe micelle. Due to the hydrophobic interior of the micelle, hydrophobicdrugs may be more readily encapsulated within the micelles. In someembodiments, the drug is hydrophobic and has low water solubility. Insome embodiments, the drug has a log p of about 2 to about 8. In someembodiments, the drug is a chemotherapeutic agent. In some embodiments,the drug is doxorubicin. In some embodiments, the drug is β-lapachone.In some embodiments, the drug is paclitaxel.

The drug may be incorporated into the micelles using methods known inthe art, such as solvent evaporation. Examples of drug incorporation maybe found in, e.g. Example 4 below. Briefly, for example, drug may beencapsulated in micelles by first dissolving the drug and the blockco-polymer in organic solution. Addition of this solution to an aqueoussolution, optionally under sonication, may result inmicelle-encapsulated drug.

Therapeutic and Diagnostic Methods

Micelles comprising a drug may be used to treat e.g. cancers,cardiovascular disease, inflammation, an autophagy-related disease, orlysosomal storage disease, or other diseases wherein the drug may bedelivered to the appropriate location due to localized pH differences(e.g. a pH different from physiological pH (7.4)). Micelles fortherapeutic methods may optionally further comprise a labeling moiety(e.g. to assist in the imaging of the treatment) and/or a targetingmoiety (e.g. to target a specific cell surface marker or to target themicelles for endocytic delivery). In some embodiments, the disordertreated is a cancer. In some embodiments, the cancer comprises a solidtumor. In embodiments wherein the micelle comprises a targeting moiety,non-solid cancers may be treated. In some embodiments, the disordertreated is lysosomal storage disease. In some embodiments, the micelleshave a pH transition value of about 6.3 to about 7.2 (e.g. for deliveryto the tumor microenvironment). In some embodiments, the micelles have apH transition value of about 5.0 to about 6.5 (e.g. for delivery tointracellular organelles). In some embodiments, the micelles have a pHtransition value of about 6.2 or above 6.2 (e.g. for delivery to earlyendosomes).

In some embodiments, the micelles have a pH transition value of about5.5 (e.g. for delivery to late endosomes or lysosomes). In someembodiments, the micelles have a pH transition value of about 6.3 toabout 6.9. In some embodiments, the micelles have a pH transition valueof about 5.0 to about 6.2. In some embodiments, the micelles have a pHtransition value of about 5.9 to about 6.2. In some embodiments, themicelles have a pH transition value of about 5.0 to about 5.5. Asdescribed in the Examples, nanoprobes 4, 3, 7 and 6 have fluorescencetransition pH values of 5.4, 6.3, 6.8 and 7.2, respectively. In someembodiments, non-targeted pHAM with higher pH_(t) (e.g. 7.2, 6.8) may beused to delivery drug to tumors. In some embodiments, targeted pHAM withlower pH_(t)(e.g. 5.4, 6.3) may be used to delivery drug to endocyticcompartments.

Micelles comprising a labeling moiety may be used in imagingapplications, for example, imaging tumors or endocytic compartments.Micelles for diagnostic methods may optionally further comprise atargeting moiety (e.g. to target a specific cell surface marker or totarget the micelles for endocytic delivery). In some embodiments, themethod is used to diagnose a tumor in the individual. In someembodiments, the method is used to monitor a tumor in the individual,for example to monitor the effects of a treatment. In some embodiments,the micelle is used for imaging early endosomal activity. In someembodiments, the micelle is used for imaging late endosomal activity. Insome embodiments, the micelle is used for imaging lysosomal activity. Insome embodiments, the micelles have a pH transition value of about 6.3to about 7.2 (e.g. for delivery to the tumor microenvironment). In someembodiments, the micelles have a pH transition value of about 5.0 toabout 6.5 (e.g. for delivery to intracellular organelles). In someembodiments, the micelles have a pH transition value of about 6.2 orabove 6.2 (e.g. for delivery to early endosomes).

In some embodiments, the micelles have a pH transition value of about5.5 (e.g. for delivery to late endosomes or lysosomes). In someembodiments, the micelles have a pH transition value of about 6.3 toabout 6.9 (e.g. for imaging the tumor microenvironment). In someembodiments, the micelles have a pH transition value of about 5.0 toabout 6.2 (e.g. for imaging intracellular organelles). In someembodiments, the micelles have a pH transition value of about 5.9 toabout 6.2 (e.g. for imaging early endosomes). In some embodiments, themicelles have a pH transition value of about 5.0 to about 5.5 (e.g. forimaging late endosomes or lysosomes). As described in the Examples,nanoprobes 4, 3, 7 and 6 have fluorescence transition pH values of 5.4,6.3, 6.8 and 7.2, respectively. In some embodiments, non-targeted pHAMwith higher pH_(t) (e.g. 7.2, 6.8) may be used for imaging tumors. Insome embodiments, targeted pHAM with lower pH_(t) (e.g. 5.4, 6.3) may beused for imaging endocytic compartments, or for imaging tumors viaendocytic uptake.

More than one type of label may be used in the compositions of theinvention. For example, different NIR fluorophores (e.g. withdistinctive excitation/emission wavelengths) may be used to generate aseries of multi-chromatic nanoprobes for different biomarkers. Thiscreates a multichromatic set of nanoprobes that allow the simultaneousimaging of several molecular targets (e.g. VEGFR2 and α_(v)β₃) which mayfurther improve the imaging efficacy of angiogenic tumor vasculature.

The invention further provides a composition comprising a micelle and apharmaceutically acceptable carrier. Such composition may beadministered to the individual by any suitable method, such as, forexample, injection (e.g. intravenous injection) or infusion.Administration may be local or systemic.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention in any manner.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. In particular,all publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

EXAMPLES

Unless indicated otherwise, temperature is in degrees Centigrade andpressure is at or near atmospheric pressure.

Example 1: Synthesis of Tunable, pH-Activatable Micellar (pHAM)Nanoparticles I. Syntheses of Methacrylate Monomers

2-(Tetramethyleneimino) ethanol (C5A), 2-(pentamethyleneimino) ethanol(C6A) and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) werepurchased from Sigma-Aldrich. 2-(Hexamethyleneimino) ethanol (C7A) and2-(dibutylamino) ethanol (DBA) were purchased from Alfa Aesar Companyand TCI America Inc., respectively. NHS-tetramethyl rhodamine (NHS-TMR)was purchased from Invitrogen Company. Monomers 2-(dimethylamino)ethylmethacrylate (DMA-MA), 2-(diethylamino)ethyl methacrylate (DEA-MA),2-(diisopropyl amino)ethyl methacrylate (DPA-MA) and 2-aminoethylmethacrylate (AMA) were purchased from Polyscience Company. AMA wasrecrystallized twice with isopropanol and ethyl acetate (3:7). PEGmacroinitiator, MeO-PEG₁₁₄-Br, was prepared from 2-bromo-2-methylpropanoyl bromide and MeO-PEG₁₁₄-OH according to the procedure inliterature (Bronstein et al., J. Phys. Chem. B, 2005, 109:18786-18798).Other solvents and reagents were used as received from Sigma-Aldrich orFisher Scientific Inc.

All new methacrylate monomers (C5A-MA, C6A-MA, C7A-MA, DBA-MA) weresynthesized following a similar method. Synthesis of2-(pentamethyleneimino)ethyl methacrylate (C6A-MA) is described as arepresentative procedure. First, 2-(pentamethyleneimino)ethanol (12.9 g,0.1 mol), triethylamine (10.1 g, 0.1 mol), and inhibitor hydroquinone(0.11 g, 0.001 mol) were dissolved in 100 mL Tetrahydrofuran (THF) andthen methacryloyl chloride (10.4 g, 0.1 mol) was added dropwise into athree-neck flask. The solution was refluxed in THF for 2 hrs. Afterreaction, the solution was filtered to remove the precipitatedtriethylamine-HCl salts, and THF solvent was removed by rotovap. Theresulting residue was distilled in vacuo (83-87° C. at 0.05 mm Hg) as acolorless liquid. After syntheses, the monomers were characterized by ¹HNMR. All the NMR spectra were obtained in CDCl₃ using tetramethylsilane(TMS) as the internal reference on a Varian 500 MHz ¹H NMR spectrometer.The characterization and yield for the monomers are as following areshown in Table 1.

TABLE 1 Characterization and yield for methacrylate monomers.Methacrylate Monomer Characterization

¹H NMR (TMS, CDCl₃, ppm): 6.09 (br, 1H, CHH═C(CH₃)—), 5.54 (br, 1H,CHH═C(CH₃)—), 4.26 (t, J = 6.2 H_(z), 2H, —OCH₂CH₂N—), 2.76 (t, J = 6.2H_(z), 2H, —OCH₂CH₂N—), 2.56 (m, 2H, —N(CH₂CH₂)₂), 1.92 (s, 3H,CH₂═C(CH₃)—), 1.73 (m, 4H, —N(CH₂CH₂)₂). Yield: 78%

¹H NMR (TMS, CDCl₃, ppm): 6.04 (br, 1H, CHH═C(CH₃)—), 5.50 (br, 1H,CHH═C(CH₃)—), 4.22 (t, J = 6.4 H_(z), 2H, —OCH₂CH₂N—), 2.60 (t, J = 6.5H_(z), 2H, —OCH₂CH₂N—), 2.40 (m, 4H, —N(CH₂CH₂)₂CH₂), 1.88 (s, 3H,CH₂═C(CH₃)—), 1.52 (m, 4H, —N(CH₂CH₂)₂CH₂), 1.36 (m, 2H,—N(CH₂CH₂)₂CH₂). Yield: 70%

¹H NMR (TMS, CDCl₃, ppm): 6.09 (br, 1H, CHH═C(CH₃)—), 5.55 (br, 1H,CHH═C(CH₃)—), 4.24 (t, J = 6.5 H_(z), 2H, —OCH₂CH₂N—), 2.84 (t, J = 6.5H_(z), 2H, —OCH₂CH₂N—), 2.72 (m, 4H, —N(CH₂CH₂CH₂)₂), 1.94 (s, 3H,CH₂═C(CH₃)—), 1.63 (m, 4H, —N(CH₂CH₂CH₂)₂), 1.58 (m, 4H,—N(CH₂CH₂CH₂)₂). Yield: 54%

¹H NMR (TMS, CDCl₃, ppm): 6.09 (br, 1H, CHH═C(CH₃)—), 5.55 (br, 1H,CHH═C(CH₃)—), 4.19 (t, J = 6.3 H_(z), 2H, —OCH₂CH₂N—), 2.73 (t, J = 6.3H_(z), 2H, —OCH₂CH₂N—), 2.46 (t, J = 7.6 H_(z), 2H, —N(CH₂CH₂CH₂CH₃)₂),1.93 (s, 3H, CH₂═C(CH₃)—), 1.41 (m, 4H, —N(CH₂CH₂CH₂CH₃)₂), 1.29 (m, 4H,—N(CH₂CH₂CH₂CH₃)₂), 0.89 (t, J = 7.3 Hz, 6H, —N(CH₂CH₂CH₂CH₃)₂). Yield:53%

II. Synthesis of PEO-b-PR and PEO-b-(PR-r-TMR) Block Copolymers

Two series of block copolymers (PEO-b-PR (y=0) and PEO-b-PR-r-TMR, FIG.1A) with different tertiary amine-containing segments (PR) andpoly(ethylene oxide) (PEO) segments were made by atom transfer radicalpolymerization (ATRP; Tsarevsky & Matyjaszewski, Chem. Rev. 2007,107:2270-2299; Ma et al., Macromolecules 2003, 36:3475-3484). In thelinear di-alkyl series (see FIG. 1B, R groups 1, 2, 3, and 4) the chainlength was varied from methyl to butyl groups; in the cyclic series (seeFIG. 1B, R groups 5, 6 and 7), the ring size from 5- to 7-membered ringswas varied.

A pH-insensitive dye, tetramethyl rhodamine (TMR; Albertazzi et al. Am.Chem. Soc. 2010, 132:18158-18167) was used as a model fluorophore andconjugated in the PR block as an imaging beacon to investigate the pHresponsive properties of pHAM nanoparticles. As described in more detailbelow, at higher pH, neutral PR segments co-operatively self-assembleinto the hydrophobic cores of micelles, which results in the aggregationof fluorophores and quenching of fluorescent signals through mechanismsof Förster resonance energy transfer between TMR molecules (homo-FRET)and photoinduced electron transfer (PeT) from tertiary amines to TMR(Kobayashi et al., Chem. Rev. 2010, 110:2620-2640; Uchiyama et al.,Chem. Int. Ed. 2008, 47:4667-4669; Lakowicz, Principles of FluorescenceSpectroscopy, 3rd ed., Springer, New York City, 2006, pp. 443-475;Diaz-Fernandez et al., Chem. Eur. J. 2006, 12:921-930). At lower pH, PRsegments become protonated and positively charged, leading to micelledisassembly and dramatic increase in fluorescence emission due to theincrease in TMR distance and decrease in PeT (FIG. 1C)).

PEO-b-PR copolymers (FIG. 2A) were first synthesized by atom transferradical polymerization (ATRP) method. The dye-free copolymers were usedin polymer characterizations and measurement of pKa and critical micelleconcentrations (Tables 2 and 3). PEO-b-PDPA is used as an example toillustrate the procedure. First, DPA-MA (1.06 g, 5 mmol), PMDETA (21 μL,0.1 mmol), and MeO-PEG₁₁₄-Br (0.5 g, 0.1 mmol) were charged into apolymerization tube. Then a mixture of 2-propanol (2 mL) and DMF (2 mL)was added to dissolve the monomer and initiator. After three cycles offreeze-pump-thaw to remove oxygen, CuBr (14 mg, 0.1 mmol) was added intothe reaction tube under nitrogen atmosphere, and the tube was sealed invacuo. The polymerization was carried out at 40° C. for 8 hrs. Afterpolymerization, the reaction mixture was diluted with 10 mL THF, andpassed through an Al₂O₃ column to remove the catalyst. The THF solventwas removed by rotovap. The residue was dialyzed in distilled water andlyophilized to obtain a white powder. The resulting PEO-b-PR copolymerswere characterized by ¹H 500 MHz NMR, gel permeation chromatography(Viscotech GPCmax, PLgel 5 μm MIXED-D columns by Polymer Labs, THF aseluent at 1 mL/min). Table 2 lists the yield, molecular weights (M. andMW) and polydispersity index (PDI) of each copolymer. PEO-b-PDPA(without labeling moiety) indicates block copolymer (3), PEO-b-PDBA(without labeling moiety) indicates block copolymer (4), PEO-b-PC6A(without labeling moiety) indicates block copolymer (6), and PEO-b-PC7A(without labeling moiety) indicates block copolymer (7).

TABLE 2 Characterization of PEO-b-PR diblock copolymers. YieldM_(w, GPC) M_(n, GPC) Repeating units M_(n,) ₁ _(H NMR) Copolymer (%)(×10⁻⁴ D)^(a) (×10⁻⁴ D)^(a) PDI^(a) in the PR block^(b) (×10⁻⁴)^(b) 1 711.47 1.36 1.08 61 1.46 2 62 1.91 1.75 1.09 58 1.57 3 71 1.14 1.04 1.1045 1.46 4 81 1.24 1.04 1.19 51 1.73 5 73 1.41 1.26 1.12 49 1.40 6 651.61 1.38 1.17 45 1.38 7 78 1.83 1.40 1.31 49 1.54 ^(a)Number-averaged(M_(n)), weight-averaged molecular weight (M_(w)) and polydispersityindex (PDI = M_(w)/M_(n)) were determined by GPC using THF as theeluent; ^(b)Determined by ¹H NMR.

To introduce the TMR dye, AMA was used in the copolymer synthesis (FIG.2B). Synthesis of PEO-b-(PR-r-AMA) copolymers followed the proceduredescribed above. Three primary amino groups were introduced into eachpolymer chain by controlling the feeding ratio of the AMA monomer to theinitiator (ratio=3). Similar yields and molecular weights were obtainedfor these PEO-b-(PR-r-AMA) copolymers. For TMR conjugation,PEO-b-(PR-r-AMA) (50 mg) was first dissolved in 2 mL DMF. Then NHS-TMRester (1.5 equivalents to the molar amount of the primary amino group)was added. The reaction mixture was stirred at room temperature for twodays. The copolymers were purified by preparative gel permeationchromatography (PLgel Prep 10 μm 10E3A 300×25 mm columns by Varian, THFas eluent at 5 mL/min) to remove the free dye molecules. The producedPEO-b-(PR-r-TMR) copolymers were lyophilized and kept at −20° C. forstorage. In the TMR-containing copolymers, the number of repeating unitsin the PR block was 70.

III. Preparation of Micelle Nanoparticles

Micelles were prepared following a solvent evaporation method aspreviously published (Nasongkla et al., Nano. Lett. 2006, 6:2427-2430).In the example of PEO-b-(PDPA-r-TMR), 24 mg of the copolymer was firstdissolved in 1 mL THF and then added into 4 mL distilled water dropwiseunder sonication. The THF was allowed to evaporate for 4 hrs by airstream. Then distilled water was added to adjust the polymerconcentration to 4 mg/mL as a stock solution. After micelle formation,the nanoparticles were characterized by transmission electron microscopy(TEM, JEOL 1200 EX model) for micelle size and morphology, dynamic lightscattering (DLS, Malvern MicroV model, He—Ne laser, λ=632 nm) forhydrodynamic diameter (Dh).

Example 2: Characterization of Tunable, pH-Activatable MicellarNanoparticles

Synthesized micellar nanoparticles were characterized to demonstratetheir pH-responsive properties both for pH response in the physiologicalrange (5.0-7.4) as well as for their temporal response.

I. Fluorescence Characterizations

In this study, conjugated TMR fluorophore was used as an imaging beaconto investigate the pH-responsive properties of pHAM nanoparticles.(Polyethylene oxide)-b-poly((dimethyl-amino)ethyl methacrylate)(PEO-b-PDMA, (1) was used as an “always ON” control where no micelles orfluorescence quenching was observed in the tested pH range (4.5-8.0) dueto the strong hydrophilicity of the PDMA block. First, fluorescenceemission spectra of pHAM nanoprobes (3, 4, 6, 7) and PEO-b-(PDMA-r-TMR)were obtained on a Hitachi fluorometer (F-7500 model). For eachcopolymer, the sample was initially prepared in MilliQ water at theconcentration of 6 mg/mL. Then the stock solution was diluted in 0.2 Mcitric-phosphate buffers with different pH values. The terminal polymerconcentration was controlled at 0.1 mg/mL. The nanoprobe was excited at545 nm, and the emission spectra were collected from 560 to 700 nm. Theemission and excitation slits were both 5 nm.

For fluorescence lifetime measurements, the fluorescence decays of TMRfrom PEO-b-(PDPA-r-TMR) (3) and PEO-b-(PDBA-r-TMR) (4) (both at 0.1mg/mL) were measured. For nanoprobe 3 (pH_(t)=6.3), the life times weremeasured at pH=7.4 and 5.5 (above and below the pH_(t), respectively) insodium phosphate/citric acid buffers. Similarly, for nanoprobe 4(pH=5.4), the life times were measured at pH=7.4 and 4.9. In bothexperiments, free TMR dye (0.005 mg/mL) was also measured as a control.These studies were carried out on a LaserStrobe fluorescence lifetimeinstrument (Photon Technology International, Inc., Birmingham, N.J.),which consists of a nitrogen laser (GL-3300) linked to a dye laser (GL302) and a stroboscopic detector. C-540A (Exciton, Inc., Dayton, Ohio)dye solution was used to generate an excitation wavelength of 540 nm.The decay curves were analyzed at the wavelength of 570 nm. The emissionmonochromator slit was at 4 nm. All measurements were conducted at roomtemperature. The IRF (instrument response function) was determined bymeasuring scattered light from a solution of glycogen. The fluorescenceintensity decay data were analyzed by the single exponential decayfunction, using the software supplied with the PTI instrument.

Fluorescent images of a series of nanoprobe solutions at different pHvalues illustrate a sharp fluorescence transition for each nanoprobe,illustrating the tunable, ultra pH responsive properties of pHAMnanoprobes.

Normalized fluorescence intensity (NFI) vs. pH curves (FIG. 3A)permitted quantitative assessment of the pH responsive properties of thepHAM nanoprobes. NFI was calculated as the ratio of[F−F_(min)]/[F_(max)−F_(min)], where F was the fluorescence intensity ofthe nanoprobe at any given pH, and F_(max) and F_(min) was the maximaland minimal fluorescence intensities at the ON/OFF states, respectively.The emission intensity at 580 nm was used to quantify the ultra-pHresponse for different pHAM nanoprobes as shown in FIG. 3A. To quantifythe sharpness in pH response, ΔpH_(10-90%), the pH range in which theNFI value varies from 10% to 90%, for all the pHAM nanoprobes wasevaluated. The sharpness values were 0.21, 0.23, 0.24, and 0.20 pH unitfor nanoprobes 4, 6, 3 and 7, respectively.

The small values of ΔpH_(10-90%) indicate a remarkable pH sensitivity asit represents a<2-fold change in proton concentration (i.e.[H⁺]_(10%)/[H⁺]_(90%)=10^(ΔpH10-90%)). In comparison, for smallmolecular dyes (Urano, et al., Nat. Med. 2009, 15:104-109), thesharpness value is about 2 pH unit (100-fold in [H⁺]) for the samedegree of emission change, consistent with Henderson-Hasselbalchequation (Atkins & De Paula, Physical Chemistry, Oxford UniversityPress, 2009). In addition to the pH sharpness, the ratio of F_(max) andF_(min) (R_(F)=F_(max)/F_(min)) was also measured to quantify thefluorescence response between the ON/OFF states. The values of R_(F)range from 10 to 55 fold (Table 3), demonstrating the large fluorescenceresponse of the nanoprobes. Consistent with the decreased emissionintensity in the micelles, the data demonstrated that excited state ofTMR had a much shorter life time (e.g. 0.44 ns for nanoprobe 3, in themicelles (pH=7.4) than the free dye (1.97 ns) at pH 7.4 or thedisassembled unimers at pH 5.5 (1.84 ns).

TABLE 3 Characterization of PEO-b-(PR-r-TMR) nanoprobes. pKa^(a) CMC^(b)D_(h) R_(F) τ_(1/2) Copolymer Monomer Polymer (mg/mL) (nm)^(c)(F_(max)/F_(min))^(d) ΔpH_(10-90%) (ms)^(e) 1 8.4 7.4 — — 1.0 — — 2 9.27.4 — — 1.8 — — 3 8.5 6.3 0.001 41 55 0.20 3.2 ± 0.1 4 6.9 5.1 0.003 4320 0.17  3.9 ± 0.1^(f) 5 9.1 7.6 — — — — — 6 8.9 6.9 0.004 39 10 0.172.7 ± 0.1 7 8.6 6.7 0.003 38 23 0.23 3.0 ± 0.2 ^(a)Determined by pHtitration experiments. ^(b)Determined by I₁/I₃ ratio of pyrene probe atpH 7.4; ^(c)Determined by DLS at copolymer concentration of 1 mg/mL andpH = 7.4; ^(d)Determined by rhodamine fluorescence emission intensity;^(e)Determined by stopped-flow measurement by mixing 20 μL 5 mg/mLpolymer solution with 80 μL phosphate buffer at pH 5.5; ^(f)pH = 4.9buffer was used to account for the low pH_(t) value of 4 (5.4).

In summary, pH-activatable micellar nanoparticles demonstrate tunabilityand ultra-sensitive pH response in the physiological range (pH 5.0-7.4),large increases in emission intensity between ON/OFF states (up to 55times), and only require <0.25 pH for activation.

II. pH Temporal Response

This study used stopped-flow measurements to gauge fluorescenceactivation in synthesized pH-activatable micellar nanoparticles.Stopped-flow measurements of pHAM nanoprobes were conducted using aBio-Logic SFM-3 instrument. All experiments were carried out at roomtemperature at different pH values in the sodium phosphate/citric acidbuffer. A monochromator was used for excitation at 540 nm and thefluorescence intensity at 570 nm long pass was recorded. Experimentswere controlled by BioKine 16 V 3.03 software and had an estimated deadtime of 1.5 ms.

The stopped-flow experiments showed that fluorescence activation wasvery fast, with most nanoprobes fully activated within 5 ms at lower pH(e.g. τ_(1/2)=3.7 ms for 4, FIG. 3B). The ultra-sensitive pH responsewas only observed with nanoprobes 4, 3, 7 and 6. The fluorescencetransition pH values (pH_(t), the pH at which F=0.5×(F_(max)+F_(min)))were 5.4, 6.3, 6.8 and 7.2 for nanoprobes 4, 3, 7 and 6, respectively(FIG. 3A). The other copolymers either did not show any pH response(e.g., 1) or only broad pH responses (e.g. 2, 5, data not shown).

In summary, the stopped-flow experiments demonstrated that thepH-activatable micellar nanoparticles have fast temporal response in therange of less than 5 ms.

III. pH Titration Curves of Copolymers and Constituent Monomers andSubsequent ¹H NMR Spectra Analysis

Without being bound to theory, it is believed that hydrophobicmicellization is the driving force of the ultra-pH responsive propertiesof pHAM, and a critical threshold of hydrophobicity in the PR segment isnecessary to achieve the co-operative response. To test this hypothesis,the pH titration curves of two representative block copolymers, 5 and 7,and their corresponding monomers were compared (FIG. 4A).

In a typical procedure, a PEO-b-PR copolymer or its correspondingmonomer was first dissolved in 0.1 N HCl to reach the finalconcentration of 5-10 mg/mL. pH titration was carried out by addingsmall volumes (50-100 μL increments) of 0.1 N NaOH solution understirring. The pH increase in the range of 2 to 11 was monitored as afunction of total added volume of NaOH (V_(NaOH)). The pH values weremeasured using a Mettler Toledo pH meter with a microelectrode. FIG. 4Ashows the representative titration curves for the cyclic PEO-b-PRcopolymers (5 and 7) and corresponding monomers. For each sample, thepKa value was calculated as the pH in the middle of the two equivalencepoints in the titration curve. The pKa values for all the PEO-b-PRcopolymers and corresponding monomers were listed in Table 3.

Both monomers behaved like small ionizable molecules with broad pHresponses over added volumes of NaOH. Copolymer 5 showed a similar broadpH response. In contrast, copolymer 7 had a dramatically sharpened pHtransition, demonstrating a greatly increased buffer capacity.Deuterated ¹H NMR spectra of 5 and 7 at different ionization states oftertiary amines ([R₃NH⁺]/[R₃N]₀) further support the hypothesis (FIG.4B). The PEO segment did not change its peak intensity and was used asan internal standard. Throughout the ionization states, the protonresonance peaks for the PR segment of 5 were easily visualized althoughthe peak intensity decreased with broadened peak width at higher pH,reflecting the bulk aggregation behavior of the copolymer. For 7,neutral state of the copolymer (i.e. 0%) led to completely suppressedresonance peaks in the PR segment due to the formation of highly compactmicelle cores. Transmission electron microscopy (TEM) of 7 in aqueoussolution demonstrated the formation of micelles above its pKa (6.7) atpH 7.4 and complete micelle dissociation at pH 5.5 (FIG. 4C). Incomparison, no micelles were formed from 5 at either pH (data notshown).

In summary, these data suggested that hydrophobic micellization was theprimary driving force for the observed cooperative deprotonationbehavior of the ammonium groups in 7.

IV. Measurement of Critical Micelle Concentration (CMC) of PEO-b-PRDiblock Copolymers

CMC of each PEO-b-PR copolymer was measured in the 0.2 M sodiumphosphate buffer at pH 7.4. First, a copolymer stock solution (3 mg/mL)was diluted to different concentrations in the same buffer. In eachsolution, 5 μL pyrene in THF solution (2×10⁻⁴ mol/L) was added to 2 mLpolymer solution to produce the final pyrene concentration at 5×10⁻⁷mol/L. The fluorescence spectra were recorded on a Hitachi fluoremeter(F-7500 model) with the excitation wavelength of 339 nm and theexcitation and emission slits at 10.0 nm and 1.0 nm, respectively. TheI₁ and I₃ values were measured as the maximum emission intensity at ca.372 and 382 nm, respectively. I₁/I₃ ratio was plotted as a function ofpolymer concentration at different pH values. I₁/I₃ ratio reflects thepolarity of the pyrene environment where partition of pyrene in thehydrophobic micelle core leads to decreased I₁/I₃ values(Kalyanasundaram et al., J. Am. Chem. Soc. 1977, 99:2039-2044; Winnik,Chem. Rev. 1993, 93:587-614). CMC values were measured as the thresholdpolymer concentration at which micelles were formed in solution. Toavoid TMR interference, PEO-b-PR copolymers without TMR conjugation wereused in these studies. The CMC values at pH 7.4 were listed in Table 3.

Example 3: Location and Mechanism of Intracellular pHAM (TMR Nanoprobes)Activation I. Confocal Laser Scanning Microscopy in Human Lung CarcinomaCells

To investigate the intracellular activations of pHAM, nanoprobe 3 inhuman H2009 lung cancer cells was examined by confocal laser scanningmicroscopy and the activation of pHAM nanoprobes in H2009 cells wasquantified by relative fluorescence intensity (FIG. 5).

H2009 cells were cultured in RPMI 1640 medium (Invitrogen, CA)supplemented with 5% fetal bovine serum (FBS), 100 IU/mL penicillin and100 μg/mL streptomycin at 37° C. in 5% CO2 atmosphere. For subcellulartrafficking and colocalization studies, H2009 cells were transfectedwith baculovirus using Organelle Lights™ Endosomes-GFP and Lysosomes-GFPBacMam 1.0 kits (Molecular Probes, OR) for Rab5a (early endosome marker)and Lamp1 (late endosome/lysosome marker) labeling, respectively. Cellswere then cultured in growth medium for further analysis. For confocalimaging studies of micelle uptake and intracellular activation, H2009cells were plated in glass bottom dishes (MatTek, MA) in 2 mL completeRPMI medium and incubated with nanoprobe 3 at a polymer concentration of0.2 mg/mL at pH 7.4. Confocal images were captured at 0, 15, 30, 45, and60 min after addition of micelles. After 60 min incubation, 0.1 N HClsolution (250 μL) was added into medium to acidify the medium pH to 5.0and cells were immediately imaged. The images were analyzed usingImage-J software. Five independent measurements were presented as themean±standard deviation. For colocalization experiments, transfectedcells expressing Rab5a-GFP or Lamp1-GFP were seeded in glass bottomdishes in 2 mL complete RPMI medium without phenol red. After 24 hr cellgrowth, 0.4 mg of nanoprobe 3 or 4 (5 mg/mL copolymer solution) in PBS(pH 7.4) was added into medium to give a final polymer concentration of0.2 mg/mL. Images were captured at designated time points by a NikonECLIPSE TE2000-E confocal microscope with a 100× objective lens. GFP andTMR were excited at 488 and 543 nm, respectively. The emissionwavelengths of GFP and TMR were 515 and 595 nm, respectively.

Because pHAM nanoprobes are “silent” at neutral pH, they were directlyapplied in the culture medium and the kinetics of their internalizationwas monitored without the need to remove the medium. Right after thenanoprobe addition, neither the H2009 cells nor the medium showedobservable fluorescence signal. At 15 min, punctuate fluorescent dotsappeared inside the cells. The number of fluorescent dots increased overtime. Signal to noise ratio of the H2009 cells (SNR_(Cell), usingfluorescence intensity at time 0 as the background noise) allowedfurther quantification of the increased nanoprobe uptake and activationover time. At 60 min, a 31-fold increase in SNR_(Cell) (2.14±0.17×10³)was observed over the medium (SNR_(Med)=69.3±9.1, P<0.001) wheremajority of the nanoprobes were still present (FIG. 5A). Then 0.1N HClsolution was added to acidify the medium pH to 5.0 and considerableincrease in fluorescence intensity in the medium background was found. Areverse trend of fluorescence contrast was observed, where SNR_(Cell)was 74% of SNR_(Med) (P<0.05) (FIG. 5B).

These data illustrated that pHAM nanoprobes can dramatically increasethe contrast sensitivity of cancer cells compared to potentially alwaysON nanoprobes (as in the case after HCl was added).

II. Activation of pHAM in Endocytic Organelles in Human Lung CancerCells

To further investigate whether different endocytic organelles canselectively activate pHAM, H2009 cells were transfected with greenfluorescent protein (GFP)-fused Rab5a and Lamp I biomarkers in earlyendosomes and late endosomes/lysosomes, respectively.

H2009 cells were plated in glass bottom dishes (MatTek, MA) in 2 mLcomplete RPMI medium and incubated with nanoprobe 3 at a polymerconcentration of 0.2 mg/mL at pH 7.4. Confocal images were captured at0, 15, 30, 45, and 60 min after addition of micelles. After 60 minincubation, 0.1 N HCl solution (250 μL) was added into medium to acidifythe medium pH to 5.0 and cells were immediately imaged. The images wereanalyzed using Image-J software. Five independent measurements werepresented as the mean±standard deviation. For colocalizationexperiments, transfected cells expressing Rab5a-GFP or Lamp1-GFP wereseeded in glass bottom dishes in 2 mL complete RPMI medium withoutphenol red. After 24 hr cell growth, 0.4 mg of nanoprobe 3 or 4 (5 mg/mLcopolymer solution) in PBS (pH 7.4) was added into medium to give afinal polymer concentration of 0.2 mg/mL. Images were captured atdesignated time points by a Nikon ECLIPSE TE2000-E confocal microscopewith a 100× objective lens. GFP and TMR were excited at 488 and 543 nm,respectively. The emission wavelengths of GFP and TMR were 515 and 595nm, respectively. For experiments on the inhibition of acidification oflysosomes with bafilomycin A1 and its effect on intracellular activationof nanoprobes 3 and 4, transfected H2009 cells expressing Lamp 1-GFP wasseeded in glass bottom dishes in 2 mL complete RPMI 1640 medium withoutphenol red. After 24 h cell growth, the medium was replaced with freshmedium containing bafilomycin A1 (final concentration=1 M) and cellswere incubated at 37° C. for 1 h. Then, 0.4 mg of nanoprobe 3 or 4 inPBS (pH 7.4) was added into medium to give a final polymer concentrationof 0.2 mg/mL. After incubation at 37° C. for 1 h, cells were imaged by aNikon ECLIPSE TE2000-E confocal microscope with a 100×objective lens.GFP and TMR were excited at 488 and 543 nm, respectively. The emissionwavelengths of GFP and TMR were 515 and 595 nm, respectively. Afterimages captured, the medium was replaced by fresh medium. The cells wereincubated at 37° C. for 5 h, followed by confocal microscopy analysis.

Two pHAM nanoprobes (3 and 4 with pH_(t) of 6.3 and 5.4, respectively)were incubated with H2009 cells and confocal imaging was used to examinethe subcellular locations for pHAM activation. H2009 cells (N=30-50)with 20 or more colocalized dots (i.e. activated pHAM within earlyendosomes or lysosomes) were identified as positive and the percentagewas quantified (FIGS. 6A and 6B). For nanoprobe 3, 80% of cells werepositive in colocalization with early endosomes at 30 min, whereas only12% colocalized with late endosomes/lysosomes. Over time, colocalizationof activated 3 decreased with early endosomes but increased with lateendosomes/lysosomes (FIG. 6A). In contrast, nanoprobe 4 (pH=5.4) showeda different pattern of subcellular location for activation. At alltimes, less than 10% of positive cells were found with early endosomecolocalization. Instead, almost all the activated nanoprobe 4colocalized with late endosomes/lysosomes (FIG. 6B). FIG. 6C and FIG. 6Ddepict the different processes of intracellular uptake and activation ofthe two nanoprobes. Nanoprobe 3 can be quickly activated inside earlyendosomes with higher vesicular pH (5.9-6.2) (Casey et al., Nat. Rev.Mol. Cell Biol. 2010, 11:50-61; Modi et al., Nat. Nanotech. 2009,4:325-330) and the activation is sustained as the nanoprobes trafficinto late endosomes/lysosomes. By contrast, nanoprobe 4 is almostexclusively activated inside the late endosomes/lysosomes with lowervesicular pH (5.0-5.5) (Casey et al., Nat. Rev. Mol. Cell Biol. 2010,11:50-61; Modi et al., Nat. Nanotech. 2009, 4:325-330). Similar resultswere also found with human SLK tumor endothelial cells (data not shown).

These data demonstrate the feasibility of targeting small differences inthe vesicular pH inside different endocytic organelles by the pHAMnanoparticles.

To verify the intracellular activation mechanism of pHAM, H2009 cellswere incubated with bafilomycin A1 for 1 hr and then added nanoprobe 3.Bafilomycin is a specific inhibitor of vacuolar-type H⁺-ATPase(V-ATPase; Gagliardi et al., Curr. Med. Chem. 1999, 6:1197-1212.), whichis responsible for the proton pumping across the plasma membranes andacidification of intracellular organelles (e.g. lysosomes). Data showthat in the presence of bafilomycin A1, nanoprobe 3 was not activated asindicated by the absence of TMR fluorescence. After removal ofbafilomycin A1 and 3 in the culture medium, the activation of 3 emergedwith colocalization of TMR fluorescence with lamp 1-GFP labeledlysosomes. Similar results were also found with nanoprobe 4 in H2009cells.

These experiments demonstrated that the synthesized nanoparticles are“silent” in the media at pH 7.4 but can be activated upon uptake intothe cells. Moreover, nanoparticles with pH transitions at 6.3 and 5.4can be selectively activated in different endocytic compartments such asearly endosomes (pH 5.9-6.2) and lysosomes (5.0-5.5). These datademonstrate the feasibility of targeting small differences in thevesicular pH inside different endocytic organelles by the pHAMnanoparticles.

Example 4: Chemotherapeutic Encapsulation into pHAM Nanoparticles

This study sought to demonstrate that pHAM nanoparticles couldencapsulate a high percentage of chemotherapeutics and subsequentlyquickly release it when exposed to an acidic environment similar to whatis observed in tumor cells.

I. Encapsulation of Doxorubicin into Micelles

PEO-b-PC6A was synthesized as above (see Example 1 (I and II)).Doxorubicin encapsulation in micelles was achieved by first dissolvingdoxorubicin and PEO-b-PC6A in water and hydrochloric acid. This solutionwas then added drop by drop into a 0.1M pH 9 buffer solution undersonication.

By using this method, doxorubicin loading percentages between 5 and 6percent out of a theoretical loading of 10 percent were obtained. Drugloading was calculated by dissolving doxorubicin-encapsulated micellesin chloroform and then measure the UV-vis absorbance.

II. Release of Doxorubicin Upon Exposure to Acidic Environments

Doxorubicin release experiments were conducted by measuring thefluorescence intensity at different time points of thedoxorubicin-loaded micelles in various pH environments. At first, 125 μLof doxorubicin-loaded micelles was mixed with 175 μL water in a cuvette,and an initial fluorescence spectrum was taken. Then, 10-20 μL of 5M pHbuffer was added to the cuvette to measure the doxorubicin release overtime. Drug concentration was calculated based on fluorescencecalibration curves of free doxorubicin in water and pH buffer. At lowconcentrations (<0.025 mg/mL), fluorescence intensity and concentrationare directly proportional. Fluorescence quenching occurs at higherconcentrations.

The release study shows that doxorubicin releases from the micellesrapidly at pH 5.0 and that the micelles at pH 7.4 are relatively stable.At pH 5.0, the micelles release doxorubicin rapidly in the first twohours and afterwards, the release is very slow. At pH 7.4, doxorubicinslowly releases out of the micelles after several hours, but themajority of the drug remains encapsulated (FIG. 7). At lowconcentrations (<0.025 mg/mL), fluorescence intensity and concentrationare directly proportional. Fluorescence quenching occurs at higherconcentrations.

This study demonstrated that polymeric micelles can encapsulate a highpercentage of doxorubicin (˜6%) and that polymers that are protonated inacidic conditions can dissociate much faster than polymers that undergohydrolysis at low pH values. Release studies showed that the micellescan release doxorubicin rapidly at pH 5.0, with the majority of the drugreleased in the first two hours.

III. Encapsulation of Paclitaxel into Micelles

Paclitaxel-loaded micelles were prepared according to a previouslypublished procedure.

In brief, 20 mg of MeO-PEO5k-PDPA25k and 2 mg of paclitaxel weredissolved in 1 mL THF. Then, the mixture was rapidly added into 10 mL ofMilli-Q water under sonication. The mixture was ultrafiltrated for morethan 6 times to remove THF using the micro-ultrafiltration system. Theresulting micellar solution was placed at room temperature for 4 hourand filtrated through a 0.45 m cellulose membrane to remove anyprecipitates in micelle solution. Paclitaxel loading content inpolymeric micelles was determined by disintegrating of micelles inacetonitrile. Paclitaxel concentration was determined by HPLC using areversed-phase Cis column (5 μm, 4.6×250 mm) with a mobile phaseconsisting of 34% acetonitrile and 66% water at 227 nm at the flow rateof 1 mL/min. Paclitaxel content in MeO-PEO5k-PDPA25k micelles was8.3±0.6%.

Example 5: Generation of a pHAM-NIR Fluorophores Comprising Cypate forTumor Angiogenesis Imaging I. Synthesis of NIR-pHAM

Cypate-NHS esters (an NIR dye) were synthesized following publishedprocedures (FIG. 8; Lopalco, et al., Org. Biomol. Chem., 2009,7:856-859; Ye et al., Bioconjug. Chem, 2007, 19:225-234). FIG. 8 shows arepresentative synthetic scheme of NIR-NHS and PEO-b-(PR-r-NIR)copolymers. Reaction of 1,2,2-trimethyl-1H-benz[e]indole (A) with3-bromopropanoic acid in 1,2-dichlorobenzene at 110° C. yielded B.Further reaction of B with malonaldehyde bis(phenylimine)monohydrochloride (n=1) or glutaconaldehyde dianil monohydrochloride(n=2) yielded the corresponding NIR fluorophores (C). Treatment of Cwith O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TSTU) and N,N-diisopropylethylamine (DIEA) in dry DMF yielded NIR-NHSester. Finally, the PEO-b-(PR-r-NIR) was synthesized through conjugationof NIR-NHS onto the block copolymers bearing the primary amino groups.In the cypate-containing copolymers, the number of repeating units inthe PR block was 70. After syntheses, polymers were fully characterizedby gel permeation chromatography, ¹H NMR, and fluorescence spectroscopy.Useful analogs with different excitation/emission wavelengths (e.g.λ_(ex)/λ_(em)=678/704 nm when n=1; λ_(ex)/λ_(em)=781/808 nm when n=2)were subsequently produced.

II. Optimization of NIR-pHAM Fluorophores

Preliminary studies on TMR-pHAM show that PR length and TMR number mayaffect the ultra-pH response. Adequate PR length may provide forcooperative micellization and directly affects pH response, i.e.ΔpH_(10-90%). The TMR density may control the fluorescence response. Forexample, an optimal F_(max)/F_(min) of 55 was achieved for TMR nanoprobe3 at y=3 without the observable intramolecular fluorescence quenching atthe ON state. In comparison, at y=1, only a F_(max)/F_(min) of 10 wasobtained (data not shown).

For the NIR-pHAM development, the PR length (70) is maintained toinvestigate the optimal NIR density per polymer chain. It is anticipatedthat different fluorophores (i.e. cypate vs. TMR) have differenthomoFRET and PET quenching effects, which may affect the optimal pHAMcomposition. To quantify the contributions from homoFRET and PET, aNIR-conjugated polymer is blended with NIR-free polymer andsystematically the molar ratios of NIR-conjugated polymer are varied.Extrapolation of quenching coefficient to one NIR dye per micellepermits the quantification of PET contribution. The cypate density (e.g.y=1, 3, 6) on the PR segment is then systematically increased. Quenchingefficiency is measured and correlated with the homoFRET model (Lakowicz(ed.), Principles of Fluorescence Spectroscopy, Edn. 3^(rd), 443-475(Springer, New York City; 2006), which is inversely proportional to r⁶(r is the distance between the dye pairs in the micelle core).

A tunable set of NIR-pHAM (i.e. nanoprobes 3, 4, 6, 7, where TMR isreplaced with NIR dye) nanoprobes with pH transitions at 5.4, 6.3, 6.8and 7.2, respectively is then synthesized. Slight variations of pH_(t)may result when a different dye (e.g. cypate) is used. After NIR-pHAMsyntheses, pH and fluorescence responses (e.g. pH_(t), ΔpH_(10-90%),F_(max)/F_(min), as described previously), article size (TEM and DLS),critical micelle concentration and fluorescence life times are measured.

Example 6: Development of Vascular-Targeted cRGDfK-pHAM

This study demonstrates the development of cRGDfK-pHAM to target pHsensitive polymeric micelle nanoparticles to the vasculature of tumors.The small peptide ligand cRGDfK (cRGD) specifically targets αvβ3integrins (CD61) which are over-expressed in angiogenic tumorendothelial cells.

Thiol-maleimide chemistry was used for ligand conjugation on the pHAMsurface (FIG. 9). MAL-PEO-b-PR was mixed with PEO-b-(PR-r-NIR) at 10 mol% molar ratio of MAL-PEO-b-PR. After micelle formation, cRGD-SH peptideswere conjugated via thiol-maleimide linkage. The peptide conjugation wasmonitored by the disappearance of maleimide group (6.7 ppm) andformation of the aromatic group (7.0-7.5 ppm) from D-Phe on the cRGDfKby the ¹H NMR. Amino acid analysis was further used to quantify thepeptide density on the surface of pHAM nanoprobes (Khemtong, et al.,Cancer Res., 2009, 69:1651-1658). TEM and DLS was used to examine theligand functionalization on the particle size and morphology, andfluorescence spectrophotometry was used to verify the pH-responsivefluorescence properties of pHAM. Laser scanning confocal microscopy wasthe primary tool to examine the kinetics of cell uptake andintracellular activation of the targeted pHAM.

Human umbilical vascular endothelial cells (HUVECs) were used in thesestudies. This cell line is well accepted as a cell culture model invitro for angiogenic endothelial cells and αvβ3 integrin isover-expressed on HUVEC cells (Ellis et al., J. Vasc. Res., 2003,40:234-243; Vag et al., Contrast Media Mol. Imaging, 2009, 4:192-198).FIG. 10 shows the contrast specificity of cRGD-encodedPEG-b-(PDPA-r-NIR) pHAM (pH transition=6.3) in HUVEC cells. The surfacedensity of cRGD/cRAD was controlled at 20 mol %. To examineαvβ3-specificity, cRAD-encoded pHAM and free cRGD block+cRGD-encodedpHAM were used as controls. HUVEC cells were cultured in EGM mediumprior to incubation of different pHAM samples (polymer concentration:0.2 mg/mL) for 3 hrs. In the cRGD block control, 20 molar excess of freecRGD peptides were co-incubated with cRGD-pHAM to compete for αvβ3binding. Because pHAM is silent at medium pH, activation of pHAM insideHUVEC cells can be directly imaged without the need to remove themedium.

The data demonstrated that after cell incubation, cRGD-pHAM showeddramatically increased fluorescence intensity inside HUVEC cells. Incomparison, cRAD-pHAM and cRGD block controls showed little fluorescencesignals. ROI analyses of different HUVEC cells showed the averagefluorescence intensity was 15.2±3.5, 1.4±0.2, 1.5±0.2 for cRGD-pHAM,cRAD-pHAM, and cRGD block control, respectively (FIG. 10A). Thefluorescence for the medium background of similar ROI size was0.56±0.09. The culture medium was used as the background noise tocalculate the contrast over noise ratio (CNR=(FIpHAM−FImed)/FImed, whereFIpHAM and FImed are the fluorescence intensity of pHAM sample andmedium, respectively) for different pHAM conditions. The values of CNRwere 26.1±6.2, 1.5±0.4, and 1.6±0.4 for cRGD-pHAM, cRAD-pHAM, and cRGDblock control, respectively (FIG. 10B). It is worth noting that >10-foldincrease in CNR for cRGD-pHAM was observed over the two controls,indicating αvβ3-specific targeting (P<0.01). In particular, thiscontrast was observed in the presence of a high concentration (0.2mg/mL) of “silent” pHAM nanoprobes in the cell culture medium.

Example 7: Evaluation of the Specificity and Efficacy of Targeted pHAMin the Imaging of Distinctive Angiogenesis Biomarkers in Tumor-BearingMice In Vivo

I. pHAM Activation in the Tumor Vasculature

Since TMR has short excitation/emission wavelengths (λex=540 nm, λem=580nm), these studies used cRGD-encoded, TMR-conjugated pHAM nanoparticlesto demonstrate pHAM activation in tumor vasculature.

Athymic nude mice bearing A549 tumor xenografts (100-200 mm³, n=3 foreach group) were used in these studies. cRGD- and cRAD-encodedPEG-b-(PDPA-r-TMR) pHAM nanoprobes were used with 20 mol % surfacedensity. Nanoprobes were injected at 14 μmol TMR/kg dose via the tailvein and animals were sacrificed 3 hrs after pHAM injection. Variousorgans were removed and placed on a Petri dish and imaged by IVISSpectrum.

TMR signals of the explanted organs and tumor tissues from cRAD-encodedand cRGD-encoded TMR-pHAM can be directly observed by a Maestrofluorescence imaging instrument with identical imaging conditions.Despite the limited tissue penetration of TMR, tumor from cRGD-encodedpHAM clearly showed higher fluorescence intensity than that fromcRAD-encoded pHAM, as well as the adjacent muscle tissues. In bothgroups, the blood drops did not show any fluorescence signals,demonstrating the intended background suppression effect of pHAM inblood. Meanwhile, liver appeared to be the major organ that took up bothpHAM formulations, consistent with the RES clearance of nanosizedparticles (Moghimi, et al., Pharmacol. Rev., 2001, 53:283-318).

After ex vivo imaging, tumor tissues were frozen and sectioned at 8 m.Confocal imaging of tumor tissues showed a remarkable increase offluorescence intensity in cRGD-pHAM treated tumor than cRAD-pHAMcontrol. To verify the location of pHAM activation, tumor sections werefirst stained with rat primary anti-mouse mAb against PECAM (CD31),followed by washing and staining with Delight 488-conjugated anti-ratsecondary antibody. Overlay images show that majority of pHAM activationco-localized with the vasculature stain, indicating the active targetingand activation of cRGD-encoded pHAM in the tumor vasculature. This studydemonstrates the feasibility of targeting specific angiogenesisbiomarkers (i.e. αvβ3) by cRGD-encoded pHAM in tumor-bearing mice. Toovercome the short tissue penetration of TMR dye, NIR-pHAM nanoprobes(e.g. cypate, λex/λem=781/808 nm when n=2) may be used for furtheranimal studies in vivo.

II. Evaluation of Targeted NIR-pHAM Nanoprobes with Optimal pH_(t)Values

The influence of pH_(t) on the imaging specificity of angiogenesisbiomarkers is investigated. In this series of studies, cRGD-encodedNIR-pHAM is used as a model system and cRAD-encoded NIR-pHAM as acontrol. NIR-conjugated PEG-b-PDBA (pH=5.4), PEG-b-PDPA (6.3), orPEO-b-PC7A (6.8) nanoprobes are evaluated. cRGD-pHAM nanoprobes withlarger pH_(t) values (e.g. 6.8) may lead to faster fluorescence responsetime inside early stage endosomes. However, it may also be moresusceptible to be “activated” by other non-α_(v)β3 related mechanisms,such as acidic pH in tumor microenvironment. Vice versa, cRGD-pHAM withlower pH_(t) values (e.g. 5.4) can be more specifically turned “ON” viaα_(v)β₃-mediated endocytosis; however, it may take longer time for themto be activated in angiogenic endothelial cells.

In this series of experiments, we inject the cRGD- and cRAD-encodedNIR-pHAMs with different pH_(t) via the tail veins of mice bearing A549tumors. The fluorescence intensity of tumors and other organs arerecorded over time to examine the kinetics of pHAM activation. LivingImage 4.0 software is used to display the 3D volume images superimposedwith mouse anatomy. For tumor tissues, the fluorescence intensity isplotted over time to examine whether saturation kinetics is present forcRGD-encoded NIR-pHAM (as expected from receptor saturation). Ifsaturation kinetics is observed, the optimal pHAM dose as the minimaldose that allows for receptor saturation is determined. This dose isthen used in subsequent studies to minimize non-specific uptake in otherorgans (e.g. liver). The CNRs of tumors over the surrounding muscletissues for cRGD-encoded vs. cRAD-encoded NIR-pHAM is then calculatedand compared to investigate the target-specific contrast due toα_(v)β₃-mediated endocytosis. For NIR-pHAMs with different pH_(t)values, CNRs between the targeted (i.e. cRGD-encoded) and non-targeted(i.e. cRAD-encoded) groups across different NIR-pHAM designs iscompared. These results are correlated with data on stealth pHAM (PEOsurface) activation in tumor microenvironment. This results of thisstudy selects the most optimal pH_(t) design for NIR-pHAM to imagespecific angiogenesis biomarkers in vivo.

cRGD-encoded NIR-pHAM (NIR-conjugated PEG-b-PDPA (pH=6.3)) was used as amodel system and non-targeted NIR-pHAM as a control. The NIR-conjugatedPEG-b-PDPA had the structure of Formula I with the following:

R′ R1/R2 n z R″ m x y L R″′ —CH₃ iPr/iPr 2 114 —CH₃ 2 70 3 Cypate BrCy5.5The targeted NIR-conjugated PEG-b-PDPA had 10 mol % surface density ofcRGD.

cRGD-encoded and non-targeted NIR-conjugated PEG-b-PDPA wereintravenously injected via the tail veins of athymic nude mice bearingA549 lung tumors. The in vivo NIR fluorescence intensity was recorded 3hours postinjection. In a comparison group, a blocking dose of cRGDfKpeptide was injected 30 min prior to the cRGD-encoded NIR-conjugatedPEG-b-PDPA administration.

The tumor from cRGD-encoded NIR-conjugated PEG-b-PDPA clearly showedhigher fluorescence intensity than that that from non-targetedNIR-conjugated PEG-b-PDPA or the cRGD blocking group.

III. Pharmacokinetics Studies of cRGD-Encoded pHAM

This study showed the blood circulation time of cRGD-encoded pHAM(targeted micelles) and cRGD-free pHAM (nontargeted micelles) in A549tumor-bearing mice.

Female athymic nude mice (20-25 g) were inoculated s.c. on the rightflank with human non-small cell lung cancer A549 cells (5×10⁶cells/mouse). Tumors were allowed to reach 200-300 mm³ before injectionof micelles. For PK studies, cRGD-free pHAM or 10% cRGD-encodedPEG-b-(PDPA-r-TMR) pHAM were injected at a dose of 20 mg/kg micellesthrough the tail vein. Blood was collected at 2 min, 3, 6, 12, 24 and 48hours after i.v. injection. Plasma was isolated from RBCs bycentrifugation at 1,000 rpm for 10 min. The plasma was stored at 4° C.for further analysis. Polymer was extracted from plasma with acidicmethanol (0.1 M HCl:MeOH, 3:7, v/v) and detected with a fluorometerusing excitation and emission wavelengths of 545 and 580 nm,respectively.

Both cRGD-encoded pHAM and cRGD-free pHAM displayed prolonged bloodcirculation time. The blood half lives of cRGD-encoded pHAM andcRGD-free pHAM were 10.0 and 9.5 hours, respectively (FIG. 11).

IV. Testing the Generality of NIR-pHAM Nanoplatform in Imaging α_(v)β₃Integrin and VEGFR2 in Several Tumor Xenograft Models.

NIR-pHAM formulation with the optimal pH_(t) is used in these studies.For the VEGFR2-targeted nanoprobes, we purify the Fab_(R2)′-SH fragmentof RAFL-1 mAb for NIR-pHAM conjugation. A non-specific Fab′-SH is alsoprepared from control rat IgG. Fab′-SH is conjugated to the NIR-pHAMsurface via thiol-maleimide chemistry. RAFL-1-NIR immuno conjugate isused as the always ON control. For the α_(v)β₃-targeted NIR-pHAM,cRGD-NIR as a small molecular dye conjugate is also synthesized. It isexpected that these always ON probes have elevated blood signals withlimited imaging payload increase at the targeted site, which havesignificantly less contrast sensitivity compared to the correspondingNIR-pHAM nanoprobes.

The targeted nanoprobes are investigated in other more clinicallyrelevant tumor models in an orthotopic MDA-MB-231 breast tumor model inthe mammary pad of female nude mice (Ran et al., Neoplasia, 2003,5:297-307) orthotopic MiaPaca-2 pancreatic tumor model in nu/nu mice(Korpanty et al., Cancer Res., 2007, 13:323-330). Both tumor modelsexpress high levels of angiogenesis biomarkers (e.g. VEGFR2, α_(v)β₃,endoglin). Imaging specificity and efficacy of NIR-pHAM nanoprobes inthese tumor models is evaluated and results are validated byimmunohistochemistry of these angiogenesis biomarkers in tissuesections.

Example 8. Evaluation of Activation of Non-Targeted NIR-pHAM in AcidicTumors

Extracellular pH is becoming an important physiological parameter tostudy tumor microenvironment and metabolism. (Cardone et al., NatureRev. Cancer, 2005, 5:786-795; Gerweck & Seetharaman, Cancer Res. 1996,56:1194-1198; Helmlinger et al., Nature Medicine, 1997, 3:177-182).Aerobic glycolysis (aka, Warburg effect), conversion of glucose tolactic acid in the presence of oxygen, is uniquely observed in cancers.To maintain a healthy intracellular pH (˜7.2), cancer cells utilizeseveral transport systems (e.g. Na⁺/H⁺ exchange, vacuolar H⁺ ATPases(V-ATPase), Na⁺/HCO₃ ⁻ exchange) to export the protons from insidecells. This results in microenvironmental acidosis that furtherfacilitates cancer invasion through ECM degradation and promotion ofangiogenesis.

Prior to studying the pHAM activation, the map in tumors is firstmeasured using MRI relaxometry method for imaging of tissue pH in vivo(Garcia-Martin et al., Magn. Reason. Med., 2006, 55:309-315; Raghunandet al., Magn. Reason. Med., 2003, 49:249-257). After measurement by MRIthe activation of non-targeted NIR-pHAM in the tumor microenvironment isevaluated. Due to the small size of pHAM (diameter 40-50 nm), theyaccumulate in the tumor interstitium through the leaky tumormicrovasculature. In a typical experiment, NIR-pHAM nanoprobes areinjected via the tail vein. 3D activation map and dynamic contrast overtime are measured on the IVIS Spectrum. Living Image (4.0) softwareprovided by the manufacturer is used to analyze the spatial and temporalactivation of NIR-pHAM nanoprobes. Moreover, the quantitative 3Dfluorescence (FLIT4) toolset is used to co-register the optical imageswith the map from MRI. The pattern of pHAM activation with the map intumors is then compared. The NIR-pHAM activation profiles are examinedand compared for nanoprobes with different pH transitions (i.e. 5.4,6.3, 6.8, 7.2).

The experiments show the following: (1) closely correlated pH_(e) andpH_(t) relationships between the tumor microenvironmental pH andNIR-pHAM activation, respectively; (2) for NIR-pHAM with high pHtransitions (i.e. 6.8 or 7.2), because of the ultra-pH response of thetested pHAM nanoprobes (i.e. <0.25 pH unit for OFF/ON transitions), theyare highly sensitive imaging probes for acidic tumors and are useful fortumor drug delivery; and (3) for NIR-pHAM with low pH transitions (i.e.5.4 or 6.3), their lack of activation by the acidic tumormicroenvironment results in achieving the imaging specificity forangiogenesis biomarkers.

PEG-PC7A-Cy5.5 nanoprobes were tested. The structure of PEG-PC7A-Cy5.5utilized had the structure of Formula I with the following:

R′ R1/R2 n z R″ m x y L R″′ —CH₃ —(CH₂)₆— 2 114 —CH₃ 2 70 3 Cypate BrCy5.5PEG-PC7A-Cy5.5 nanoprobes (pH_(t)=6.7) were intravenously injected (25mg/kg) via the tail vein of athymic nude mice bearing A549 lung tumors.In the comparison group, α-cyano-4-hydroxycinnamate, a monocarboxylatetransferase 1 (MCT1) inhibitor, was injected 24 hours prior to nanoprobeadministration. The tumor from the non-targeted PEG-PC7A-Cy5.5nanoprobes clearly showed higher fluorescence intensity than that fromthe MCT1 inhibitor group.

Example 9: Development of VEGFR2-Targeted pHAM

This study demonstrates the development of Fab_(R2)′-functionalizedmicelles for specific targeting of VEGFR2 receptors on the surface ofendothelial cells. The Fab′ fragment of RAFL-1 mAb is used for specifictargeting to VEGFR2 receptors since VEGFR2 is over-expressed inangiogenic tumor endothelial cells. RAFL-1 mAb binds to VEGFR2 with highaffinity (15 μM) and specificity (Ran et al., Neoplasia, 2003,5:297-307) and, following purification of the Fab_(R2)′-SH fragment ofRAFL-1 for surface functionalization, Fab_(R2)′-functionalized liposomesshowed >30-fold increase in cell uptake in mouse endothelial cells overthe control liposomes (Marconescu, PhD. Thesis, UT Southwestern MedicalCenter, Dallas, 2008). Compared with the whole mAb, Fab_(R2)′-SH has theadvantage of introducing a smaller targeting moiety (50 vs. 150 kD), andsuperior presentation of binding epitope on the pHAM surface (i.e.facing solution instead of random orientation for whole mAb).

Thiol-maleimide chemistry is used for ligand conjugation on the pHAMsurface. MAL-PEO-b-PR is mixed with PEO-b-(PR-r-NIR) at different molarratios (e.g. 20 mol % of MAL-PEO-b-PR). For each pHAM copolymer, itscorresponding maleimide-terminated copolymer is then synthesized (FIG.8). After micelle formation, Fab_(R2)′-SH peptides are conjugated viathiol-maleimide linkage. Amino acid analysis is further used to quantifythe peptide density on the surface of pHAM nanoprobes (Khemtong, et al.,Cancer Res., 2009, 69:1651-1658). TEM and DLS is used to examine theligand functionalization on the particle size and morphology, andfluorescence spectrophotometry is used to verify the pH-responsivefluorescence properties of pHAM. Laser scanning confocal microscopy isthe primary tool to examine the kinetics of cell uptake andintracellular activation of the targeted pHAM.

1.-93. (canceled)
 94. An in vitro method for delivery of a drug to lateor early endosomes or lysosomes, comprising administration of thecomposition to an individual in need thereof, the composition comprisinga pH sensitive micelle comprising a block copolymer comprisingpoly(ethylene oxide) (PEO), and a hydrophobic polymer segment comprising

wherein R′ is —H or —CH₃, wherein R is —NR¹R², wherein R¹ and R² arestraight chain alkyl groups, wherein R¹ and R² are the same ordifferent, wherein R¹ and R² together have from 5 to 16 carbons, whereinR¹ and R² may optionally join to form a ring, wherein n is 1 to about10, and wherein x is about 20 to about 200 in total.
 95. The method ofclaim 94, wherein the endosome is an early stage endosome.
 96. Themethod of claim 94, wherein the endosome is a late stage endosome. 97.The method of claim 94, wherein the micelle has a pH transition value ofabout 5.9 to about 6.5.
 98. The method of claim 94, wherein the micellehas a pH transition value of about 5.0 to about 5.5.
 99. The method ofclaim 94, wherein the micelle has a pH transition range of less thanabout 1.0 unit.
 100. The method of claim 94, wherein the micelle has apH transition range of less than about 0.5 unit.
 101. The method ofclaim 94, wherein R¹ and R² join to form a ring.
 102. The method ofclaim 94, wherein R¹ and R² are the same.
 103. The method of claim 94,wherein R¹ and R² are different
 104. The method of claim 94, wherein R¹and R² each have 3 to 8 carbons.
 105. The method of claim 94, wherein R¹and R² are n-butyl.
 106. The method of claim 94, wherein R¹ and R²together are —(CH₂)₅— or —(CH₂)₆—.
 107. The method of claim 94, whereinn is 1 to
 4. 108. The method of claim 94, wherein x is about 40 to about100 in total.
 109. The method of claim 94, wherein the compositionfurther comprises a labeling moiety.